Manipulation of pyrophosphate fructose 6-phosphate 1

Manipulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase activity in

sugarcane

by Jan-Hendrik Groenewald

Dissertation presented for the degree of Doctor of Philosophy

at Stellenbosch University

April 2006 Promoter: Prof FC Botha

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DECLARATION

I, the undersigned, hereby declare that the work contained in this dissertation is my own original work and that I have not previously in its entirety or in part submitted it at any university for a degree.

19 December 2005

J-H Groenewald Date

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SUMMARY The main aim of the work presented in this thesis was to elucidate the apparent role of

pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose accumulation

in sugarcane. PFP activity in sugarcane internodal tissue is inversely correlated to the

sucrose content and positively to the water-insoluble component across varieties which

differ in their capacities to accumulate sucrose. This apparent well defined and important

role of PFP seems to stand in contrast to the ambiguity regarding PFP’s role in the general

literature as well as the results of various transgenic studies where neither the down-

regulation nor the over-expression of PFP activity had a major influence on the phenotype

of transgenic potato and tobacco plants. Based on this it was therefore thought that either

the kinetic properties of sugarcane PFP is significantly different than that of other plant

PFPs or that PFP’s role in sucrose accumulating tissues is different from that in starch

accumulating tissues.

In the first part of the study sugarcane PFP was therefore purified and its molecular and

kinetic properties were determined. It consisted of two subunits which aggregated in

dimeric, tetrameric and octameric forms depending on the presence of Fru 2,6-P2. Both the

glycolytic and gluconeogenic reactions had broad pH optima and the kinetic parameters for

all the substrates were comparable to that of other plant PFPs. The conclusion was therefore

that sugarcane PFP’s molecular and kinetic characteristics do not differ significantly from

that of other plant PFPs.

The only direct way to confirm if PFP is involved in sucrose accumulation in sugarcane is

to alter its levels in the same genetic background through genetic engineering. This was

therefore the second focus of this study. PFP activity was successfully down-regulated in

sugarcane. The transgenic plants showed no visible phenotype under greenhouse and field

conditions and sucrose concentrations in their immature internodes were significantly

increased. PFP activity was inversely correlated with sucrose content in the immature

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internodes of the transgenic lines. Both the immature and mature internodes of the

transgenic plants had significantly higher fibre contents.

This study suggests that PFP plays a significant role in glycolytic carbon flux in immature,

metabolically active sugarcane internodal tissues. The data presented here confirm that PFP

can indeed have an influence on the rate of glycolysis and carbon partitioning in these

tissues. It also implies that there are no differences between the functions of PFP in starch

and sucrose storing tissues and it supports the hypothesis that PFP provides additional

glycolytic capacity to PFK at times of high metabolic flux in biosynthetically active tissue.

This work will serve as a basis to refine future genetic manipulation strategies and could

make a valuable contribution to the productivity of South African sugarcane varieties.

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OPSOMMING

Die hoofdoelwit van die werk wat in hierdie proefskrif beskryf word, was om die potensiële

rol wat pirofosfaat fruktose 6-fosfaat 1-fosfotransferase (PFP) in sukrose akkumulering in

suikerriet mag speel, te ontrafel. PFP-aktiwiteit in suikerriet-internodale-weefsel is

omgekeerd eweredig aan die sukrose-inhoud en direk eweredig aan die wateronoplosbare

komponent in talle variëteite wat verskillende kapasiteite het om sukrose te akkumuleer.

Hierdie blykbaar duidelike en goed gedefinieerde rol van PFP staan in kontras teenoor die

onsekerheid aangaande die rol vir PFP soos wat dit in die literatuur beskryf word. Resultate

van verskeie transgeniese studies het ook getoon dat geen aansienlike fenotipiese

veranderinge deur die afregulering of die ooruitdrukking van PFP-aktiwiteit in transgeniese

aartappel- en tabakplante teweeg gebring is nie. Daar is dus op grond van hierdie inligting

afgelei dat die kinetiese eienskappe van suikerriet-PFP óf aansienlik van dié van ander

plant-PFPs verskil óf dat PFP se funksie in sukrose-akkumulerende weefsel verskil van dié

in stysel-akkumulerende weefsel.

In die eerste deel van die studie is PFP gesuiwer en die molekulêre- en kinetiese eienskappe

daarvan is bepaal. Die ensiem is uit twee subeenhede saamgestel wat in dimeriese-,

tetrameriese- of oktameriese vorme kan aggregeer, afhangend van die teenwoordigheid van

Fru 2,6-P2. Beide die glikolitiese- en die glukoneogeniese reaksies het ‘n breë pH-optimum

en die kinetiese parameters vir alle substrate het met ander plant-PFPs ooreengestem. Die

resultate het dus bevestig dat die molekulêre- en kinetiese eienskappe van suikerriet-PFP

nie aansienlik van dié van ander plant-PFPs verskil nie.

Die enigste direkte manier om die betrokkenheid van PFP by sukrose-akkumulering in

suikerriet te bevestig, is om die ensiemvlakke in presies dieselfde genetiese agtergrond,

dmv genetiese manipulering, te verander. Hierdie manipulering was dan die tweede fokus

van die studie. PFP-aktiwiteit is suksesvol afgereguleer in suikerriet. Die transgeniese

plante het nie enige sigbare fenotipe onder glashuis- of veldtoetstande getoon nie en die

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sukrose-konsentrasies in die jong internodes was aansienlik verhoog. PFP-aktiwiteit was

omgekeerd eweredig aan sukrose-inhoud in die jong internodes van transgeniese lyne. Die

veselinhoud van beide die jong- en volwasse internodes van die transgeniese plante was

aansienlik verhoog.

Resultate wat in hierdie studie verkry is, dui daarop dat PFP ‘n baie belangrike rol in

glikolitiese koolstoffluks in jong, metabolies-aktiewe suikerriet internodale weefsel speel.

Die data bevestig verder dat PFP wel die glikolise-tempo en koolstofverdeling in hierdie

weefsels beïnvloed en dat daar geen verskille tussen die funksie van PFP in sukrose- en

styselstorende weefsel is nie. Dit ondersteun dus die hipotese dat PFP bydra tot die

glikolitiese kapasiteit ten tye van hoë metaboliese fluks in biosinteties-aktiewe weefsel.

Hierdie werk vorm ‘n basis waarvandaan toekomstige genetiese manipuleringstrategieë

verfyn kan word en kan ‘n baie belangrike bydrae tot die produktiwiteit van Suid-

Afrikaanse suikerrietvariëteite lewer.

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Vir Sarita, Marko en Tian – want julle gee betekenis aan alles.

“If you stood on the bottom rail of a bridge, and leant over, and watched the river slipping slowly away beneath you, you would suddenly know everything that there is to be known.”

Winnie the Pooh on knowledge

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ACKNOWLEGEMENTS

The work described here and the dissertation itself would not have been possible without the valuable contributions of many people and institutions. I would therefore like to thank…

Frikkie, your contribution to this work is obvious and your support and hard work is very much appreciated. What I appreciate most, though, is what you taught me that can’t be captured in the pages of this book – thank you.

Sarita, not only for motivating, pleading and threatening but also for proof reading, criticising and improving what I wrote. Without your support it would not have been possible, without your love it would not have been worth while.

All my friends at the IPB who helped and supported me in so many ways.

Everyone at SASRI who helped with the collection and preparation of samples and managed the field trial. Without Barbara’s and Sandy’s help specifically this would have been a much thinner book.

SASRI, THRIP and Stellenbosch University for financial and other support.

I’m truly blessed to have the support and friendship of so many people - thanks!

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PREFACE This dissertation is presented as a compilation of six chapters. In Chapter 1 the overarching aim and approach to the study is introduced and the aims and outcomes of each individual chapter is summarised. Similarly, Chapter 6 concludes the work with a general discussion which aims to integrate the work presented in all the other chapters and focuses on the general conclusions. Chapter 2 is a literature review in which, after critical discussion of the available literature, two probable mechanisms through which PFP can influence sucrose accumulation is presented. Each of the experimental chapters (Chapter 3-5) has a distinct aim and outcome and is introduced separately. Each chapter that will be submitted for publication is written according to the style of the particular journal as listed below; these papers will be co-authored by FC Botha.

Chapter 1 General introduction.

Will not be submitted for publication. Style: Plant Physiology.

Chapter 2 Literature review: Characteristics and potential function of pyrophosphate: fructose-6-phosphate 1-phosphotransferase with special reference to the sucrose accumulation phenotype of sugarcane culm.

Plant physiology and biochemistry.

Chapter 3 Purification and characterisation of pyrophosphate fructose-6-phosphate 1-phosphotransferase from sugarcane.

Journal of plant physiology.

Chapter 4 Development and characterisation of transgenic systems for the manipulation of PFP activity in sugarcane.


Chapter 5 Down-regulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase activity in sugarcane enhances sucrose accumulation in immature internodes.

Transgenic research.

Chapter 6 General Discussion and Conclusions.


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TABLE OF CONTENTS

Content Page

Chapter 1, General introduction 1

References 5

Chapter 2, Characteristics and potential function of pyrophosphate: fructose-6-phosphate 1-phosphotransferase with special reference to the sucrose accumulation phenotype of sugarcane culm

Abstract 8

Introduction 8

Catalytic activity of PFP 11

Molecular characteristics of PFP 12

Kinetic and regulatory characteristics 14

The influence of pH on activity 15

Substrate/product interactions 16

Activation by Fru 2,6-P2 17

PFP’s role in metabolism 18

Conclusion 25

References 25

Chapter 3, Purification and characterisation of pyrophosphate: fructose-6-phosphate 1-phosphotransferase from sugarcane

Abstract 37

Introduction 38

Materials and methods 40

Results 43

Purification and molecular characterization 43

Kinetic and regulatory characterisation 47

Discussion 49

PFP activity in different sugarcane tissues 49

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Purification and molecular properties 49

Kinetic and regulatory properties 51

Implications to the sucrose accumulation phenotype 52

References 54 Chapter 4, Development and characterisation of transgenic systems for the manipulation of PFP activity in sugarcane

Abstract 60

Introduction 60


Results and discussion 65

Selection of non-plant PFPs 65

Isolation and characterisation of the PFP gene sequences 66

Expression, purification and kinetic characterisation of non-plant PFPs

68

Construction of plant expression vectors 72

Conclusion 73

References 73

Chapter 5, Down-regulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase activity in sugarcane enhances sucrose accumulation in immature internodes

Abstract 78

Introduction 79


Results and discussion 85

Molecular characterisation of transgenic plants 85

Reduction in PFP activity 86

Influence of reduced PFP activity on sugar yields in greenhouse grown plants

88

Correlation between PFP activity and sucrose yields 90

Influence of reduced PFP activity on sugar yields in field grown plants

91

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Conclusion 96

References 97

Chapter 6, General discussion and conclusions 100

References 105

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LIST OF FIGURES AND TABLES

Reference Title Page

Chapter 1

Figure 1 Interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate. While PFK and FBPase catalyses irreversible reactions in the glycolytic and gluconeogenic directions respectively, PFP catalyses a freely reversible reaction

2

Chapter 2

Figure 1 A summary of the most important reactions in sucrose metabolism in the sink tissues of sugarcane

10

Table 1 The molecular properties of selected plant PFPs 13

Table 2 Kinetic parameters of selected plant PFPs. 15

Chapter 3

Figure 1 PFP activity in various sugarcane tissue types as determined in crude extracts

44

Figure 2 SDS-PAGE and immunoblot analysis of purified sugarcane PFP

46

Figure 3 Gel filtration chromatography of purified sugarcane PFP 47

Figure 4 pH dependence of sugarcane PFP activity 48

Table 1 Kinetic parameters for the forward reaction of partially purified sugarcane PFP from internodal and callus tissue

44

Table 2 Purification of PFP from sugarcane callus 45

Table 3 Kinetic and regulatory properties of sugarcane PFP at saturating substrate concentrations

48

Chapter 4

Figure 1 Coding sequence and 3’ UTR of the sugarcane PFP-β gene 67

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Figure 2 Activity of recombinant G. lamblia and P. freudenreichii PFP in crude extracts from bacterial expression systems

69

Figure 3 pH dependence of G. lamblia and P. freudenreichii PFP activity

70

Figure 4 Influence of Fru 2,6-P2 on the activity of G. lamblia and P. freudenreichii PFP activity

70

Figure 5 Protein blot analysis using thirty-eight day serum raised against the GST fusions of G. lamblia and P. freudenreichii PFP

71

Figure 6 Schematic maps of two of the plant expression vectors constructed for the genetic manipulation of PFP activity in sugarcane

72

Table 1 Comparison of plant and non-plant PFPs 66

Table 2 Primers used to amplify a 248 bp fragment of sugarcane PFP-β

66

Table 3 Primer sequences used to amplify the P. freudenreichii PFP gene from gDNA

68

Table 4 Kinetic properties of G. lamblia and P. freudenreichii PFP compared to the metabolite concentrations in sugarcane culm

70

Table 5 Properties of the plant expression vectors constructed to manipulate PFP expression in transgenic sugarcane

72

Chapter 5

Figure 1 Northern blot analysis confirming the expression if the PFP-β transgene

86

Figure 2 PFP activity in maturing internodal tissue 87

Figure 3 PFP activity in different internodal tissues of wild type and five representative transgenic genotypes

87

Figure 4 Sugar concentrations in internode 3-5 (A) and 12-14 (B) tissue expressed as a function of fresh weight

88

Figure 5 Sugar concentrations in internode 3-5 (A) and 12-14 (B) tissue expressed as a function of dry weight

90

Figure 6 The relationship between sucrose yields and PFP activity in transgenic and control sugarcane lines

91

Figure 7 Sugar data for field grown wild type (WT) and transgenic sugarcane lines with reduced PFP activity

92

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Figure 8 Fibre content of field grown wild type (WT) and transgenic lines with reduced PFP activity

95

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ABBREVIATIONS

2,4-D 2,4-dichlorophenoxy acetic acid

ATP adenosine 5’-triphosphate

bp base pairs

BSA bovine serum albumin

CaMV-35S Cauliflower mosaic virus’ 35S ribosomal subunit’s promoter sequence

DEPC diethyl pyrocarbonate

DTT 1,4-dithiothreitol

EDTA ethylenediaminetetraacetic acid

e.g. for example

Fru fructose

Fru 6-P fructose 6-phosphate

Fru 1,6-P2 fructose 1,6-bisphosphate

Fru 2,6-P2 fructose 2,6-bisphosphate

FBPase fructose-1,6-bisphosphatase (EC 3.1.3.11)

FW fresh weight

Glc glucose

HEPES N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid

IGEPAL Polyoxyethylene nonyl phenol

kb kilo base pairs

kDa kilo Dalton

Km substrate concentration producing half maximal velocity

Ki kinetic inhibition constant

MES 2(N-morpholino) ethanesulphonic acid

MS Murashige and Skoog, i.e. Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiology Plantarum 15:473-497

µM micromolar (10-6M)

mM milimolar (10-3M)

nM nanomolar (10-9M)

PAGE polyacrylamide gel electrophoresis

PCR polymerase chain reaction

PEG polyethylene glycol

PFP pyrophosphate: fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90)

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PFK ATP: fructose 6-phosphate 1-phosphotransferase (EC 2.7.1.11)

Pi inorganic phosphate

PIPES Piperazine-1,4-bis(2-ethanesulfonic acid)

PPi inorganic pyrophosphate

PVPP polyvinil polypyrrolidone

RNAi RNA interference

SDS sodium dodecyl sulphate

SPS sucrose phosphate synthase (EC 2.4.1.14)

SuSy sucrose synthase (EC 2.4.1.13)

UDPGlc uridine 5’-diphosphoglucose

UDPGlc-DH uridine 5’-diphosphoglucose dehydrogenase

VPPase vacuolar H+-translocating inorganic pyrophosphatase (EC 3.6.1.1)

x g times gravitational force

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CHAPTER 1

General introduction

Sugarcane is one of the most valuable agricultural crops in South Africa and cane sugar,

i.e. sucrose, is the main product that is derived from it. Cane sugar generates an annual

income of approximately R6 billion and contribute an estimated R2 billion to the

country’s foreign exchange earnings (www.sasa.org.za). Approximately 350,000 South

Africans are directly or indirectly employed by the sugarcane industry and this

translates into more than a million people being dependent on the industry. To stay

competitive in a global commodity market prone to overproduction, the South African

industry has to focus on more cost effective production systems. Increasing the sucrose

concentration in commercial sugarcane varieties will be one of the most important

factors contributing towards improved cost effectiveness. The emphasis should fall here

on increased sucrose yield per ton cane and not only on an increase in tons sucrose per

unit area. For this reason sucrose storage was identified recently as the most important

research priority for the industry (Inman-Bamber et al. 2005).

Commercial sugarcane varieties are interspecific hybrids that are capable of storing

sucrose up to 62% of their dry weight or 25% of their fresh weight (Bull and Glasziou

1963, Welbaum and Meinzer 1990). Historically, increases in sucrose yield have been

accomplished through conventional breeding programs. Variety improvement through

breeding is estimated to have increased sucrose yield by 1-1.5% per annum over the last

half of the 20th century in Australia (Chapman 1996). However, these increases were

attained mainly via improvements in cane yield and not in sucrose content (Jackson

2005). In addition, there are ample suggestions that sugarcane is approaching a yield

plateau (Moore 2005 and reference therein). A possible reason for this might be that the

natural genetic potential for sucrose production has been exhausted (Grof and Campbell

2001). Genetic engineering therefore represents an opportunity to add to this potential

by the specific manipulation of endogenous genetic traits or by introducing desired

genetic traits from exogenous sources.

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Sucrose accumulation involves a multitude of metabolic and physical processes in the

cells and tissues that are involved in sucrose synthesis, transport and storage. Cytosolic

sucrose metabolism in the storage parenchyma is one of these processes that might

influence sucrose accumulation, particularly in the way it governs carbon partitioning in

these cells (Whittaker and Botha 1997, Bindon and Botha 2002). Pyrophosphate:

fructose 6-phosphate 1-phosphotransferase (PFP), in combination with ATP: fructose 6-

phosphate 1-phosphotransferase (PFK) and fructose 1,6-bisphosphatase (FBPase), plays

a central role in cytosolic carbon metabolism, representing the first committed catalytic

step towards respiration (Figure 1). PFP catalysis the reversible conversion of fructose

6-phosphate (Fru-6-P) and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fru-1,6-

P2) and inorganic phosphate (Pi) (Carnal and Black 1979). This is thought to be a near-

equilibrium reaction and is therefore able to respond to cellular metabolism in a very

flexible manner (Edwards and ap Rees 1986).

Figure 1. Interconversion of fructose 6-phosphate and fructose 1,6-bisphosphate. While PFK and FBPase catalyses irreversible reactions in the glycolytic and gluconeogenic directions respectively, PFP catalyses a freely reversible reaction.

Results from transgenic tobacco and potato plants in which PFP activity has been

reduced by much as 97% suggest that the investigated tissues either have a huge excess

of PFP and/or complementary activity or that PFP does not play a crucial role in plant

metabolism (Hajirezaei et al. 1994, Paul et al. 1995, Nielsen and Stitt 2001). Similarly,

the over-expression of non-regulated PFP activity in tobacco plants did not cause

Fructose 6-phosphate

Fructose 1,6-bisphosphate

ATP-phosphofructokinase (PFK)

ATP

ADP

PPi

Pi

PPi-phosphofructokinase (PFP)

fructose-1,6-bisphosphatase (FBPase)

glycolysis

gluconeogenesis

Fructose 6-phosphate

Fructose 1,6-bisphosphate


ATP

ADP

PPi

Pi


fructose-1,6-bisphosphatase (FBPase)

glycolysis

gluconeogenesis

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dramatic phenotypic or physiological effects either (Wood et al. 2002a, 2002b). In all

these examples the levels of PFP’s substrates/products were influenced as could be

expected of a net glycolytic reaction but all other changes in metabolite levels were of a

transient nature, disappearing as the tissues mature.

In contrast to this apparently insignificant role of PFP in these specific plants/tissues the

changes in its activity (Botha and Botha 1991, Murley et al. 1998, Krook et al. 2000)

and the subtle regulation of its activator, fructose 2,6-phosphate (Paz et al. 1985, Van

Praag and Agosti 1997, Van Praag et al. 1997), during the various stages of normal

growth and development and under changing environmental conditions suggest a more

prominent role for PFP in carbohydrate metabolism. Similarly, it also seems to play an

important role in sucrose accumulation in sugarcane. PFP activity is inversely correlated

to the sucrose content and positively to the water-insoluble component in maturing

sugarcane internodal tissues (Whittaker and Botha 1999). In addition, a significant

amount of carbon is cycled between the triose-phosphate and hexose-phosphate pools,

for which PFP is at least partially responsible (Whittaker 1997, Bindon and Botha

2002). The extent of this cycling is also inversely correlated to sucrose accumulation

across varieties (Whittaker 1997) and maturing internodal tissue (Bindon and Botha

2002). The available data therefore suggest that PFP plays an important role in carbon

partitioning in sugarcane storage tissues. Consequently, reduced PFP activity might

directly decrease glycolytic carbon flow and also reduce the extent of the triose-

phosphate : hexose-phosphate cycle, resulting in increased sucrose synthesis and/or

accumulation.

The main aim of this study was therefore to investigate this potential role of PFP in

sucrose accumulation in sugarcane. This was done by firstly characterising the

sugarcane enzyme to determine whether it has significantly different properties to other

plant PFPs, which could explain its apparent role in sucrose accumulation. Secondly,

PFP activity was down-regulated in transgenic plants to determine whether the inverse

correlation between its activity and sucrose content could be demonstrated in this direct

manner.

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To conclude, an overview of all the aims and outcomes of this study is presented in

context of the various chapters in which they were dealt with.

Chapter 2: Characteristics and potential function of pyrophosphate: fructose-6-

phosphate 1-phosphotransferase with special reference to the sucrose accumulation

phenotype of sugarcane culm.

Aim: To present the background of this study in the format of a review paper that

includes a critical discussion on the apparent role of PFP in sucrose accumulation in

sugarcane internodal tissues.

Outcomes: An overview of the molecular, kinetic and regulatory characteristics of a

representative sample of plant PFPs is presented. The potential roles of PFP in sink

tissues are discussed with specific reference to the sucrose accumulation phenotype in

sugarcane and two hypotheses are presented that might explain PFP’s role in sucrose

accumulation in sugarcane.

Chapter 3: Purification and characterisation of pyrophosphate: fructose-6-phosphate 1-

phosphotransferase from sugarcane.

Aim: To determine the molecular and kinetic properties of sugarcane PFP in order to

assist in elucidating its apparent role in sucrose accumulation.

Outcomes: Sugarcane PFP was purified to homogeneity and its molecular and kinetic

parameters were determined for the first time. No significant differences between

sugarcane and other plant PFPs were found and it was shown that the apparent

correlation between sucrose content and PFP activity is probably linked to the

genetically determined amount of activity present in the tissue and not to fine regulatory

mechanisms.

Chapter 4: Development and characterisation of transgenic systems for the

manipulation of PFP activity in sugarcane.

Aim: To establish various transformation systems that could be used to up- and down-

regulate PFP activity in sugarcane.

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Outcomes: Three gene sequences, i.e. the sugarcane PFP-β, Giardia lamblia and

Propionibacterium PFP genes, were cloned and used for the construction of six plant

expression vectors that can be used for the constitutive or phloem specific up- or down-

regulation of PFP activity in sugarcane. In addition, the two heterologous PFP proteins

were expressed, purified and characterised to confirm their bio-activity and potential

properties under in vivo conditions. Finally, antisera were raised against the two purified

proteins to enable the easy characterisation of transgenic plants.

Chapter 5: Down-regulation of Pyrophosphate: fructose 6-phosphate 1-

phosphotransferase activity in sugarcane enhances sucrose accumulation in immature

internodes.

Aim: To verify the potential role of PFP in sucrose metabolism in sugarcane and in

particular its apparent direct influence on sucrose accumulation.

Outcomes: PFP activity was successfully down-regulated in several transgenic

sugarcane lines using antisense and co-suppression constructs of the sugarcane PFP-β

gene. Reduced PFP activity significantly increased sucrose concentrations in immature,

metabolically active internodal tissues but no significant differences were apparent in

mature tissues. The data presented here support the suggested role of PFP as a bypass to

PFK at times of high metabolic flux in biosynthetically active tissues.

Chapter 6: General discussion and conclusions.

Aim: To integrate the observations and discussions of the experimental chapters.

Outcomes: An overarching conclusion regarding the role of PFP in sucrose

accumulation in sugarcane is presented and the potential focus of future research on this

topic is discussed.

REFERENCES

Bindon KA, Botha FC (2002) Carbon allocation to the insoluble fraction, respiration and triose-phosphate cycling in the sugarcane culm. Physiologia Plantarum 116: 12-19

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Botha A-M, Botha FC (1991) Pyrophosphate dependent phosphofructokinase of Citrullus lanatus: molecular forms and expression of subunits, Plant Physiol 96: 1185-1192

Bull TA, Glasziou KT (1963) The evolutionary significance of sugar accumulation in saccharum. Aust J Biol Sci 16: 737-742

Carnal NW, Black CC (1979) Pyrophosphate-dependent 6-phosphofructokinase, a new glycolytic enzyme in pineapple leaves. Biochem Biophys Res Comm 86: 20-26

Chapman LS (1996) Increase in sugar yield from plant breeding from 1946 to 1994. In DM Wilson, JA Hogarth, JA Campbell, AL Garside, eds Sugarcane: Research towards efficient and sustainable production. CSIRO, Division of tropical crops and pastures, Brisbane, pp 37-38

Edwards J, ap Rees T (1986) Sucrose partitioning in developing embryos of round and wrinkled varieties of Pisum sativum. Phytochemistry 25: 2027-2032

Grof CPL, Campbell JA (2001) Sugarcane sucrose metabolism: scope for molecular manipulation. Aust J Plant Physiol 28: 1-12

Hajirezaei M, Sonnewald U, Viola R, Carlisle S, Dennis DT, Stitt M (1994) Transgenic potato plants with strongly decreased expression of pyrophosphate: fructose-6-phosphate phosphotransferase show no visible phenotype and only minor changes in metabolic fluxes in their tubers. Planta 192: 16-30

Inman-Bamber NG, Bonnett GD, Smith DM, Thorburn PJ (2005) Sugarcane physiology: Integrating from cell to crop to advance sugarcane production. Field Crop Research 92: 115-117

Jackson PA (2005) Breeding for improved sugar content in sugarcane. Field Crop Research 92: 277-290

Krook J, Van't Slot KAE, Vruegdenhil D, Dijkema C, Van der Plas L (2000) The triose-hexose phosphate cycle and the sucrose cycle in carrot (Daucus carota L.) cell suspensions are controlled by respiration and PPi:Fructose-6-phosphate phosphotransferase. Plant Physiol 156: 595-604

Moore PH (2005) Integration of sucrose accumulation processes across hierarchical scales: towards developing an understanding of the gene-to-crop continuum. Field Crop Research 92: 119-135

Murley VR, Theodorou ME, Plaxton WC (1998) Phosphate starvation-inducible pyrophosphate-dependent phosphofructokinase occurs in plants whose roots do not form sybiotic associations with mycorrhizal fungi. Physiologia Plantarum 103: 405-414

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Nielsen TH, Stitt M (2001) Tobacco transformants with strongly decreased expression of pyrophosphate:fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes. Planta 214: 106-116

Paul M, Sonnewald U, Hajirezaei M, Dennis D, Stitt M (1995) Transgenic tobacco plants with strongly decreased expression of pyrophosphate: fructose-6-phosphate 1-phosphotransferase do not differ significantly from wild type in photosynthate partitioning, plant growth or their ability to cope with limiting phosphate, limiting nitrogen and suboptimal temperatures. Planta 196: 277-283

Paz N, Xu DP, Black CC (1985) Rapid oscillations of Fru 2,6-P2 levels in plant tissues. Plant Physiol 79: 1133-1136.

Van Praag E, Agosti RD (1997) Response of fructose-2,6-bisphosphate to environmental changes. Effect of low temperature in winter and summer wheat. Archs Sci 50: 207-215

Van Praag E, Monod D, Greppin H, Agosti RD (1997) Response of the carbohydrate metabolism and fructose-2,6-bisphosphate to environmental changes. Effects of different light treatments. Bot Helv 106: 103-112

Welbaum GE, Meinzer FC (1990) Compartmentation of solutes and water in developing sugarcane stalk tissue. Plant Physiol 93: 1147-1153

Whittaker A (1997) Pyrophosphate dependent phosphofructokinase (PFP) activity and other aspects of sucrose metabolism in sugarcane internodal tissues. PhD thesis, University of Natal, South Africa, pp 1-187

Whittaker A, Botha FC (1997) Carbon partitioning during sucrose accumulation in sugarcane internodal tissue. Plant Physiol 115: 1651-1659

Whittaker A, Botha FC (1999) Pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase activity patterns in relation to sucrose storage across sugarcane varieties. Physiologia Plantarum 107: 379-386

Wood SM, Newcomb W, Dennis DT (2002a) Overexpression of the glycolytic enzyme Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) in developing transgenic tobacco seeds results in alterations in the onset and extent of storage lipid deposition. Can J Bot 80: 993-1001

Wood SM, King SP, Kuzma MM, Blakeley SD, Newcomb W, Dennis DT (2002b) Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase overexpression in transgenic tobacco: physiological and biochemical analysis of source and sink tissues. Can J Bot 80: 983-992

- 8 -

CHAPTER 2

Characteristics and potential function of pyrophosphate: fructose-6-phosphate 1-

phosphotransferase with special reference to the sucrose accumulation phenotype

of sugarcane culm ∗

ABSTRACT

Despite the apparent ubiquity of pyrophosphate: fructose 6-phosphate 1-

phosphotransferase (PFP) in plants no clear physiological role has emerged for it.

Transgenic plants with up- or down-regulated PFP activity showed only small changes

in the levels of metabolites directly associated with it but no significant, stable

phenotypic changes. In sugarcane PFP activity is inversely correlated to sucrose content

in maturing internodal tissues, suggesting a more prominent role for it in carbohydrate

partitioning in these tissues. In this review I will therefore first give an overview of the

molecular, kinetic and regulatory characteristics of plant PFPs, which could help

elucidate its apparent role in sucrose metabolism and then I will discuss these potential

roles with specific reference to the sucrose accumulation phenotype in sugarcane.

Finally, I present two hypotheses that might explain PFP’s role in sucrose accumulation

in sugarcane.

INTRODUCTION

Sugar metabolism has been studied extensively in sugarcane in an attempt to elucidate

the molecular basis of the sucrose storing phenotype (Batta et al. 1995, Moore 1995,

Botha et al. 1996, Moore and Maretzki 1996, Lingle 1999, Moore 2005, Rae et al. 2005

and the references in these). In doing so, many of the enzyme reactions involved in

sucrose metabolism in sugarcane sink tissues (Figure 1) have been characterised. These

include sucrose phosphate synthase (SPS, Grof et al. 1998, Botha and Black 2000),

sucrose phosphatase (Gutierrez-Miceli et al. 2002), sucrose synthase (SuSy, Lingle and

∗ To be submitted to Plant Physiology and Biochemistry

- 9 -

Irvine 1994, Lingle and Dyer 2001, Schäfer et al. 2004a, 2004b and 2005), the

invertases (Zhu et al. 1997, Echeverria 1998, Vorster and Botha 1999, Rose and Botha

2000, Bosch and Botha 2004), hexo- and fructokinases (Hoepfner and Botha 2003 and

2004), ATP: fructose 6-phosphate 1-phosphotransferase (PFK, Whittaker and Botha

1999) and pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP, Whittaker

and Botha 1999). Of all these enzymes only PFP activity shows a consistent inverse

correlation with sucrose concentrations across commercial varieties and within a

segregating F1 population (Whittaker and Botha 1999).

This apparent important role of PFP in determining carbon flux in sugarcane sink

tissues is in contrast to evidence from transgenic tobacco and potato in which PFP

activity was reduced by much as 97%. Although there was a marked reduction in 3-

phosphoglycerate (3-PGA) and PEP and an increase in Fru 2,6-P2 levels in these plants,

there was no significant effect on fluxes or growth and morphology (Hajirezaei et al.

1994, Paul et al. 1995, Nielsen and Stitt 2001). These findings led the authors to

conclude that the investigated tissues either have a huge excess of PFP and/or

complementary activity or that PFP does not play a crucial role in metabolism. More

recently, the over-expression of non-regulated PFP activity in tobacco plants also did

not cause dramatic phenotypic or physiological effects but did result in a decrease in

starch accumulation in both source and sink tissues and an increase in the total lipid

content of seeds (Wood et al. 2002a, 2002b). In addition, the onset of lipid deposition

was advanced by up to 48 hours in the developing transgenic embryos (Wood et al.

2002b).

- 10 -

Figure 1. A summary of the most important reactions in sucrose metabolism in the sink tissues of sugarcane. Potential symplastic loading and the diffusion of sugars across membranes are not indicated in the diagram.

Vacuole

triose phosphate isomerase

glucose

glucose-1-phosphate

glucose-6-phosphate

fructose-6-phosphate

fructose-1,6-bisphosphate

dihydroxyacetone-P glyceraldehyde-3-P

UDP glucose pyrophosphorylase

glucose-6-phosphate isomerase


fructose-1,6-bisphosphate aldolasefructose-1,6-bisphosphate aldolase

PPi ATP

UTP

ADPPifructose-1,6-bisphosphatase (FBPase)

hexose kinase

UTP(NTP)

UDP(NDP)


phosphoglucomutase

PFK2/FBPase2fructose-2,6-bisphosphate

Sucrose

sucrose-6-phosphate

UDP-glucose fructose

sucrose synthase (SuSy) neutral invertase

H2O

sucrose phosphatase

sucrose phosphate synthase (SPS)

UDP

Pi

PPi

UDP

UDP-glucose dehydrogenase

UDP-glucoronate

CELL WALLS

Cytosol

SucroseSucrose

glucosefructose

Apoplast

soluble acid invertase

PPi 2x PiH+

H+

H+ trans-vac-PPase

glucosefructose

cell walll invertase

H+

ATP ADP2H+

2H+

H+ trans-vac-ATPase

~10%

RESPIRATION

PROTEINS

Vacuole

triose phosphate isomerase

glucose

glucose-1-phosphate

glucose-6-phosphate

fructose-6-phosphate

fructose-1,6-bisphosphate

dihydroxyacetone-P glyceraldehyde-3-P

UDP glucose pyrophosphorylase

glucose-6-phosphate isomerase


fructose-1,6-bisphosphate aldolasefructose-1,6-bisphosphate aldolase

PPi ATP

UTP

ADPPifructose-1,6-bisphosphatase (FBPase)

hexose kinase

UTP(NTP)

UDP(NDP)


phosphoglucomutase

PFK2/FBPase2fructose-2,6-bisphosphate

Sucrose

sucrose-6-phosphate

UDP-glucose fructose

sucrose synthase (SuSy) neutral invertase

H2O

sucrose phosphatase

sucrose phosphate synthase (SPS)

UDP

Pi

PPi

UDP

UDP-glucose dehydrogenase

UDP-glucoronate

CELL WALLS

Cytosol

SucroseSucrose

glucosefructose

Apoplast

soluble acid invertase

PPi 2x PiH+

H+

H+ trans-vac-PPase

glucosefructose

cell walll invertase

H+

ATP ADP2H+

2H+

H+ trans-vac-ATPase

~10%

RESPIRATION

PROTEINS

RESPIRATION

PROTEINS

- 11 -

The apparent role of PFP in sucrose accumulation in sugarcane therefore still requires

confirmation and explanation. In this review I will firstly describe the molecular, kinetic

and regulatory characteristics of PFP from other plants that might contribute to our

understanding of its role in metabolism and secondly discuss these potential roles. In the

discussion I will specifically refer to the sucrose accumulation phenotype in sugarcane

and present two hypotheses that might explain PFP’s role in sucrose accumulation.

CATALYTIC ACTIVITY OF PFP

PFP (EC 2.7.1.90) catalyses the reversible conversion of fructose 6-phosphate (Fru 6-P)

and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fru 1,6-P2) and inorganic

phosphate (Pi)(Figure 1). The enzyme was first isolated from the lower eukaryote

Entamoeba histolytica (Reeves et al. 1974) and later also from a limited number of

prokaryotes and lower eukaryotes such as Propionibacterium (O’Brien et al. 1975),

Rhodospirillum (Pfleiderer and Klemme 1980) and Giardia lamblia (Rozario et al.

1995). The first plant PFP was isolated from pineapple leaves by Carnal and Black

(1979) and is now considered to be ubiquitous in plants (Stitt 1990).

Plant PFP is a strictly cytosolic enzyme and is one of three enzymes involved in the

interconversion of Fru 6-P and Fru 1,6-P2 in this cellular compartment. The other two

being PFK (EC 2.7.1.11) and fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11)

(Figure 1). In contrast to PFP, PFK and FBPase catalyse irreversible reactions in the

glycolytic (Fru 1,6-P2 forming) and gluconeogenic (Fru 6-P forming) directions

respectively. In addition, ATP is used as the phosphoryl donor in the PFK catalysed

reaction. In most non-photosynthetic tissues the interrelationship between these three

enzymes is further convoluted by the absence of detectable levels of FBPase activity

(Entwistle and ap Rees 1990, Hatzfeld and Stitt 1990, Fernie et al. 2001), implying that

PFP is responsible for all gluconeogenic carbon flux through this step in these tissues.

In non-photosynthetic tissues where FBPase activity is present, the levels of activity are

often inadequate to sustain gluconeogenic flux (Botha and Botha 1993a, Focks and

Benning 1998), although the kinetic properties of grapefruit juice sac vesicle FBPase

suggest that it might play an important gluconeogenic role (Van Praag 1997b).

- 12 -

PFP is thought to catalyse a near-equilibrium reaction in vivo (Keq = 3.3, calculated for

the glycolytic direction) and is therefore able to respond to cellular metabolism in a very

flexible manner because it can catalyse a net flux of carbon in either the glycolytic or

gluconeogenic direction (Edwards and ap Rees 1986, Weiner et al. 1987). Its activity is

strongly modulated by metabolites such as fructose 2,6-bisphosphate (Fru 2,6-P2), Fru

6-P, Fru 1,6-P2, PPi and Pi (Cséke et al. 1982, Van Schaftingen et al. 1982, Kombrink

et al. 1984, Stitt 1989, Montavon and Kruger 1992, Nielsen and Wischmann 1995,

Theodorou and Plaxton 1996, Fernie et al. 2001) and its activity varies according to

developmental stage and environmental conditions (Botha and Botha 1991a, Hajirezaei

and Stitt 1991, Botha and Botha 1993b, Murley et al. 1998, Whittaker and Botha 1999,

Krook et al. 2000).

MOLECULAR CHARACTERISTICS OF PFP

Most plant PFPs are multimeric enzymes, composed of two immunologically distinct

peptides, namely the α- and β-subunits. The respective molecular weights of the α- and

β-subunits are approximately 66 and 60 kDa (Table 1) and these subunits can be

aggregated in di-, tetra- or octameric arrangements. The relative amounts of the two

subunits and the specific composition of the various holoenzymes can vary depending

on factors such as the developmental stage of the tissue and specific environmental

conditions and can play a role in the regulation of enzyme activity (Kruger and Dennis

1987, Botha and Botha 1991a, 1991b, Theodorou et al. 1992, Theodorou and Plaxton

1996). In addition, it has been shown that the two subunit genes are differentially

expressed in germinating castor seeds (Blakeley et al. 1992) and that isoforms of the

enzyme exist (Yan and Tao 1984, Wong et al. 1990, Botha and Botha 1993b). Active

PFP isoforms consisting of only the β-subunit has also been isolated and characterised

(Table 1). These enzymes can either be exclusively present in the specific plant, e.g.

pineapple (Trípodi and Podestá 1997), or as one of several isoforms with distinctive

kinetic properties, e.g. wheat and tomato (Yan and Tao 1984, Wong et al. 1990).

- 13 -

Table 1. The molecular properties of selected plant PFPs.

Source Molecular weight (kDa)

Plant Tissue Holoenzyme α-subunit β-subunit

α:β

ratio Reference

Banana Ripe fruit 490 (octamer) 66 60 1:1 Turner and Plaxton 2003

Barley Seedlings 500 (+20mM PPi, octamer), 240 (-PPi, tetramer)

65 60 1:1 Nielsen 1994

Carrot Tap root 294 (tetramer) 61a 59a 1:1 Wong et al. 1988

Castor bean

Germinating seeds

n.a. 67 60 0.1:1 to 0.6:1

Blakely et al. 1992

C. lanatus Cotyledons n.a. (tetramer, α-β-dimer, β-dimer )

68 65 4.8:1 to 0.8:1

Botha and Botha 1991a

Mustard Suspension cultures

520 (octamer) 66 60 1:1 Theodorou and Plaxton 1996

Pea Cotyledons 12.7S (+Fru 2,6-P2), 6.3S (-Fru 2,6-P2)

n.a.b n.a. n.a. Wu et al. 1984

Pineapple Leaves 97.2 (homodimer) - 61.5 0:1 Trípodi and Podestá 1997

Potato Tuber 265 (tetramer), 129.6 (+20mM PPi, dimer)

65 60 1:1 Kruger and Dennis 1987

Rice Seeds 103 (monomer) - - - Enomoto et al. 1992

Spinach Leaves 242 (+Fru 2,6-P2, tetramer), 165 (-Fru 2,6-P2, dimer)

n.a. n.a. n.a. Balogh et al. 1984

Tomato Fruit 443 (“oligomer”), 68 (β-dimer) and 68 (β-monomer)

66 60 1:1 or 0:1 Wong et al. 1990

Wheat Seedlings 234 (tetramer), 60 (β-dimer) 67 60 1:1 or 0:1 Yan and Tao 1984

Wheat Endosperm 170 (dimer) 90 80 n.a. Mahajan and Singh 1989 a Subunits not immunologically distinct. b n.a. = data not available.

The quaternary structure of PFP can also be influenced in vitro by the enzyme’s

interaction with metabolites such as PPi and Fru 2,6-P2. Several authors reported the

dissociation of the heterotetramer in the presence of PPi (Wu et al. 1983, Balogh et al.

1984, Kruger and Dennis 1987). Fru 2,6-P2 can prevent this dissociation and also

mediates the reassociation of the lower molecular weight, dimeric forms. In contrast,

Nielsen (1994) could only isolate an octameric form in the presence of 20 mM PPi. This

holoenzyme dissociated into tetramers in the absence of PPi and Fru 2,6-P2 had no

effect on the elution profile during gel filtration. Carrot PFP’s state of aggregation on

the other hand is insensitive to either the presence or absence of these metabolites

(Wong et al. 1988). Although it has been proposed that the aggregation state of the

enzyme can provide a mechanism by which Fru 2,6-P2 activation can favour the

glycolytic reaction (Wu et al. 1983, Wu et al. 1984, Black et al. 1985), the evidence is

- 14 -

inconclusive (Stitt 1990). In addition, activation does not necessarily lead to changes in

the molecular mass of PFP (Bertagnolli et al. 1986, MacDonald and Preiss 1986, Wong

et al. 1988).

The β-subunit has been identified as the catalytic subunit while the α-subunit is

involved in the regulation of enzyme activity through Fru 2,6-P2 (Yan and Tao 1984,

Wong et al. 1988, 1990, Carlisle et al. 1990, Cheng and Tao 1990, Botha and Botha

1993b). Accordingly, although isoforms containing only β-subunits are still activated by

Fru 2,6-P2, PFP isoforms containing the α-subunit have lower Ka values, i.e. have a

higher affinity for Fru 2,6-P2 (Yan and Tao 1984, Wong et al. 1988, 1990). Theodorou

et al. (1992) also found that induction of PFP activity under Pi starvation was due to the

de novo synthesis of the α-subunit, leading to a significant enhancement in activation by

Fru 2,6-P2. Likewise, a computer model developed for grapefruit PFP supports the

involvement of the α-subunit in the regulation of activity through Fru 2,6-P2 (Van Praag

1997a). The model further suggests that Fru 2,6-P2 can only bind when both the α- and

β-subunits are present. In contrast, the homodimeric (β2) pineapple PFP has a high

affinity for Fru 2,6-P2 compared to heteromeric PFPs (Table 2) and increase activity

more than 2-fold at optimum pH values (Trípodi and Podestá 1997).

KINETIC AND REGULATORY CHARACTERISTICS OF PFP

The kinetic and regulatory properties of various plant PFPs have been studied in detail

in an attempt to shed more light on the physiological relevance of the enzyme (Table 2).

Unfortunately, the integration and interpretation of the available kinetic data is difficult

because (i) the data were obtained under optimum conditions that do not necessarily

represent in vivo conditions, (ii) the reaction conditions used by various researchers vary

and (iii) PFP’s activity is affected by various metabolites and other buffer components,

including its substrates/products, which will inevitably be reflected in the data obtained.

Only selected kinetic and regulatory characteristics, which have clear physiological

implications, will therefore be discussed under three headings, i.e. the influence of pH

on activity, substrate/product interactions and activation by Fru 2,6-P2.

- 15 -

Table 2. Kinetic parameters of selected plant PFPs.

Glycolytic Gluconeogenic

Km a Ka Km a Ka Plant (tissue) Fru 6-P (µM) PPi (µM) Fru 2,6-P2

(nM) Fru 1,6-P2

(µM) Pi (µM) Fru 2,6-P2 (nM)

pH optimum b Reference

Banana (ripe fruits)

32 9.7 8 25 410 n.a.c f: 7.1 Turner and Plaxton 2003

Barley (seedlings)

200 8 2.8 11 480 60 n.a. Nielsen 1994

Carrot (tap root)d

430 19 n.a. 200 2300 n.a. n.a. Wong et al. 1988

Castor bean (endosperm)

300 15 10-123 23 630 60-300 f:7.3-7.7, r:7.75

Kombrink et al. 1984

Cucumber (seeds)

180 12.9 35-100 74.9 480.4 n.a. f:7.5-8.0, r:7.5-8.0

Botha et al. 1986

Grapefruit (juice sac)

159 33 6.7 61 700 n.a. n.a. Van Praag 1997a

Mustard (cell suspension)

50 15 15-4750 9 250 49 f:6.5-7.2, r:6.7-7.7

Theodorou and Plaxton 1996

Pineapple (leaves)

890 11 26.3-43.5 94 149 2.4-2.7 f:7.7, r:6.6-8.4

Trípodi and Podestá 1997

Tomato (fruit)d

380-600 20-40 4-13 40-70 610-950 n.a. n.a. Wong et al. 1990

Wheat 322 31 n.a. 139 129 n.a. 7.5 Mahajan and Singh 1989

a Km values in presence of saturating Fru 2,6-P2. b f = forward reaction (glycolytic) and r = reverse reaction (gluconeogenic) in the presence of Fru 2,6-P2. c n.a. = data not available. d Fru 2,6-P2 reduces or have little effect on affinity for Fru 6-P.

The influence of pH on activity

The pH dependence for both the forward and reverse reactions is similar and PFP

usually has a relatively broad activity range with optimum activity between 6.5 and 8.0

(Yan and Tao 1984, Kombrink et al. 1984, Botha et al. 1986, Nielsen 1994, Theodorou

and Plaxton 1996, Trípodi and Podestá 1997). The most important effect of pH is

probably its role in the activation of PFP by Fru 2,6-P2. Fully activated PFP can be less

sensitive to changes in pH than the non-activated enzyme; i.e. the extent to which Fru

2,6-P2 activates PFP will be greater at non-optimum pH values (Yan and Tao 1984,

Kombrink et al.1984, Theodorou and Plaxton 1996, Trípodi and Podestá 1997). For

particularly the glycolytic reaction, this can also be interpreted as a shift, or at least an

extension, of the optimum pH towards more acidic pH values under fully activated

conditions (Yan and Tao 1984, Kombrink et al.1984, Enomoto et al. 1992, Theodorou

- 16 -

and Plaxton 1996). This suggests a glycolytic role for these PFPs under conditions that

will induce a decrease in cytosolic pH, e.g. anoxia (Dancer and ap Rees 1989).

Exceptions to this are, for example, cucumber and wheat PFP where Fru 2,6-P2 does not

change the pH dependence and the maximum activation effect of Fru 2,6-P2 is at the

optimum pH values (Botha et al. 1986, Mahajan and Singh 1989).

Substrate/product interactions

PFP requires a bivalent cation and has the highest affinity for Mg2+ (Kombrink et al.

1984, Botha et al. 1986). Various other cations have been tested of which only Mn2+ and

Co2+ can replace Mg2+, but at lower efficiencies. In addition, PFP’s affinity for Mg2+ is

increased in the presence of Fru 2,6-P2, and Mg2+ concentrations in excess of 1mM

inhibit PFP activity in the glycolytic direction (Kombrink et al. 1984, Montavon and

Kruger 1992). More recent studies indicated that the Mg2+ cation is complexed with PPi

before it is used as the substrate in the glycolytic reaction (Montavon and Kruger 1992,

Trípodi and Podestá 1997). Moreover, PFP uses free Fru 6-P and MgPPi in the

glycolytic reaction and free Pi, free Fru 1,6-P2 and Mg2+ in the gluconeogenic reaction.

The use of the MgPPi complex is significant because more than 98% of the PPi will be

chelated in vivo (Trípodi and Podestá 1997).

PFP exhibits hyperbolic kinetics for all its substrates in both the glycolytic and

gluconeogenic reactions and in the presence or absence of Fru 2,6-P2. In the glycolytic

reaction each of the substrates, Fru 6-P and PPi, decreases the affinity of the other

substrate with increasing concentrations, i.e. the Km of Fru 6-P increases slightly with

increasing concentrations of PPi and vice versa (Stitt 1989). The same holds true for Fru

1,6-P2’s effect on the Km of Pi in the gluconeogenic reaction, but in contrast the Km of

Fru 1,6-P2 is significantly increased by Pi (Stitt 1989). Moreover, Pi also significantly

decreases PFP’s affinity for Fru 6-P and PPi in the glycolytic reaction. This inhibitory

behaviour of Pi for different plant PFPs has been characterised as either the mixed

(Kombrink et al. 1984, Enomoto et al. 1992) or noncompetitive (Botha et al. 1986, Stitt

1989) type with respect to both Fru 6-P and PPi. In contrast to Fru 6-P, increasing Pi

concentrations also strongly decrease PFP’s affinity for Fru 2,6-P2, which effectively

- 17 -

prevents the activation of PFP and results in a parallel inhibition of both the glycolytic

and gluconeogenic reactions (Kombrink and Kruger 1984, Botha et al. 1986, Mahajan

and Singh 1989, Stitt 1989, Theodorou and Plaxton 1996). Although Pi increases the Ka

for Fru 2,6-P2 in both the directions its inhibitory effect is more pronounced in the

glycolytic direction.

PPi is a powerful product inhibitor of the gluconeogenic reaction and Fru 2,6-P2 cannot

relieve this effect (Stitt 1989). It is a competitive inhibitor with respect to Fru 1,6-P2 and

a non-competitive inhibitor with respect to Pi (Stitt 1989). Finally, Fru 1,6-P2 has been

shown to act as an allosteric activator of PFP (Sabularse and Anderson 1981, Nielsen

1995), but it is unlikely to be an effective activator in vivo because of the reduced

affinity of the enzyme under physiological conditions (Theodorou and Kruger 2001).

The kinetic properties of PFP therefore strongly suggest that its activity is tightly

regulated in vivo. This regulation is mediated not only by PFP’s substrates and products

but also by Fru 2,6-P2.

Activation by Fru 2,6-P2

Fru 2,6-P2 is a potent activator of PFP. It activates the glycolytic reaction by increasing

Vmax and the enzyme’s affinity for Fru 6-P (Sabularse and Anderson 1981, Van

Schaftingen et al. 1982, Botha et al. 1986, Theodorou and Plaxton 1996). The effect on

the Km of PPi is not as clear and can vary from a decrease to a slight increase (Van

Schaftingen et al. 1982, Kombrink et al. 1984, Bertagnolli et al. 1986, Botha et al.

1986). The gluconeogenic reaction is also activated through an increase in Vmax and a

decrease in the Km of Fru 1,6-P2 (Van Schaftingen et al. 1982, Kombrink et al. 1984,

Bertagnolli et al. 1986, Botha et al. 1986, Theodorou and Plaxton 1996). PFP’s affinity

for Pi may be slightly increased, not influenced or decreased (Kombrink et al. 1984,

Botha et al. 1986, Theodorou and Plaxton 1996). Fru 2,6-P2 can alleviate the inhibitory

effect of PPi on the gluconeogenic reaction to some extent (Sabularse and Andeson

1981, Van Schaftingen et al. 1982). Both Fru 6-P and Fru 1,6-P2 increase PFP’s affinity

for Fru 2,6-P2 (Van Schaftingen et al. 1982, Kombrink et al. 1984) and Pi decreases its

affinity (Kombrink and Kruger 1984, Botha et al. 1986, Mahajan and Singh 1989, Stitt

- 18 -

1989). Based on Fru 2,6-P2’s in vitro Ka (nM range, Van Schaftingen et al. 1982,

Kombrink et al. 1984) and its estimated in vivo concentrations (µM range, Cséke et al.

1982, Scott and Kruger 1994) it was initially thought that PFP is always fully activated

in vivo. Nielsen and Wischmann (1995) suggested, however, that the concentration of

PFP subunits might be higher than the Fru 2,6-P2 concentration in some tissues,

resulting in non-activated PFP even when the concentration of Fru 2,6-P2 exceeds its Ka

by several orders of magnitude. More recently it was also shown that the inhibition of

Fru 2,6-P2 binding by physiological levels of Pi and phosphorylated intermediates can

decrease the affinity of PFP to the extent that the enzyme is sensitive to the changes in

Fru 2,6-P2 concentrations in vivo (Theodorou and Kruger 2001, Turner and Plaxton

2003). This implies that the activation state of PFP at specific Fru 2,6-P2 concentrations

can vary continuously due to changes in the enzyme’s affinity for this effector.

Although the same concentration of Fru 2,6-P2 will activate the glycolytic reaction more

than the gluconeogenic reaction (Kombrink et al. 1984, Nielsen 1994, Turner and

Plaxton 2003) only a single Fru 2,6-P2 binding site is present, which means that the

glycolytic and gluconeogenic reactions are always activated symmetrically (Stitt and

Vasella 1988, Stitt 1990, Nielsen 1994). Activation by Fru 2,6-P2 is therefore not able to

influence the direction of carbon flux directly, but would rather determine the rate by

which equilibrium is restored. Although PFP catalyses a net glycolytic reaction and

increasing Fru 2,6-P2 levels are often associated with conditions under which the rate of

glycolysis is stimulated, it is not surprising that this correlation is not absolute (Van

Schaftingen and Hers 1983, ap Rees et al. 1985a, Stitt et al. 1986, Stitt 1990, Hatzfeld

and Stitt 1991).

PFP’S ROLE IN METABOLISM

Theoretically PFP has only one “function”; to facilitate the establishment of an

equilibrium between [Fru 6-P][PPi] and [Fru 1,6-P2][Pi]. Not withstanding, many

authors suggest that despite numerous molecular and kinetic studies its exact

physiological role is still elusive (Stitt 1989, Trípodi and Podestá 1997, Murley et al.

1998, Stitt 1998, Fernie et al. 2001). Although its ubiquity, tight regulation in vivo and

- 19 -

spatial and temporal specificity suggest that it plays a critical role in plant metabolism

its apparent redundancy, as suggested by studies on transgenic plants with a reduction of

up to 97% in PFP activity (Hajirezaei et al. 1994, Paul et al. 1995, Nielsen and Stitt

2001), contributes to this ambiguity. Here we would like to argue that PFP does not

have a single, “definitive” physiological role but that its significance or “role” rather

emanates from two of its distinct (in comparison to the other enzymes catalysing the

same reactions) characteristics, i.e. (i) its ability to catalyse a reversible reaction and (ii)

by serving as a link between carbohydrate and PPi metabolism. In other words, PFP has

the ability to play various “roles” within the scope of these two traits, depending on the

specific physiological conditions.

To illustrate, PFP has been implicated in the following roles; the regulation of cytosolic

PPi concentrations (Simcox et al. 1979, ap Rees et al. 1985b, ap Rees 1988, Dancer and

ap Rees 1989, Claassen et al. 1991), the equilibration of the hexose- and triose-

phosphate pools (Dennis and Greyson 1987, Hatzfeld et al. 1990, Hajirezaei et al.

1994), the synthesis of PPi, which is required for the breakdown of sucrose through the

sucrose synthase pathway (Huber and Akazawa 1986, Black et al. 1987, ap Rees 1988),

providing a bypass to PFK at times of high metabolic flux in biosynthetically active

tissue (Dennis and Greyson 1987), regulating gluconeogenic carbon flow when FBPase

activity is low or absent (Botha and Botha 1993a, Focks and Benning 1998), relieving

stress during periods of phosphate limitation or starvation by providing an adenylate

bypass for glycolysis (Duff et al. 1989, Theodorou et al. 1992, Murley et al. 1998) and

being the preferred glycolytic path during spells of anaerobiosis and anoxia (Mertens

1991, Kato-Noguchi 2002). In all these suggested roles the two distinct traits of PFP, as

mentioned above, are relevant and will confer some advantage to the system in

comparison to alternative reactions – if available at all. Specific examples include the

ability to use PPi as phosphoryl donor during adenylate stress, gluconeogenic carbon

flux in the absence of FBPase activity and perceptive responsiveness to metabolite

levels because of the reversibility of the reaction. Based on these arguments two

different hypotheses that explain the apparent role of PFP in the sucrose accumulation

phenotype in sugarcane are proposed; the first is based on the direct influence the PFP-

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catalysed reaction can have on carbon flux and the second on the indirect influence it

can have on PPi metabolism.

Hypothesis 1: PFP plays an important role in determining carbon partitioning between

the hexose-phosphate pool and total respiratory flux in sugarcane internodal tissues.

As mentioned earlier, PFP activity in internodal sugarcane tissue is inversely correlated

to sucrose content across varieties with different sucrose yielding capacities (Whittaker

and Botha 1999). These authors further showed that although PFP activity varied

significantly between these varieties their PFK activities were very similar. In addition,

it was demonstrated that the low sucrose storing varieties allocate a significantly higher

proportion of carbon to their total respiratory pool, i.e. CO2 production and anabolic

biosynthesis, in similar tissue types (Whittaker and Botha 1997). It is therefore

reasonable to conclude that the majority of this “additional” respiratory flux in the low

sucrose storing varieties is catalysed by PFP. Reduced PFP activity under these

circumstances should therefore lead to a reduction in respiratory flux, which could

increase the availability of the precursors for sucrose synthesis. The potential impact of

reduced PFP activity on carbon flux in sugarcane should also be seen in the light of the

very high ratio of PFP:PFK activity in low sucrose storing sugarcane varieties compared

to high yielding varieties. This ratio is for example 2.1:1 for US6656-15, a low sucrose

yielding variety, and 0.9:1 for N24, a high sucrose yielding variety (Whittaker and

Botha 1999).

Testing this hypothesis against the available transgenic data is complicated by the

variety of tissues involved, i.e. photosynthetic vs. non-photosynthetic, sink vs. source

and different levels of metabolic activity. Although the silencing studies suggest that

these specific tissues are (eventually) able to compensate for the large decrease in PFP

activity by the allosteric activation of the remaining PFP and/or by the activation of

PFK (Hajirezaei et al. 1994, Paul et al. 1995, Nielsen and Stitt 2001), more transient

aspects of metabolism are influenced significantly. For example, although the final

sucrose concentration in mature transgenic tubers is unchanged, the flux into sucrose in

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the growing (metabolically active) tubers is 13 times higher when compared to the wild

type (Hajirezaei et al. 1994). It most probably indicates that PFP’s contribution to

glycolytic carbon flux is crucial at times of high metabolic flux in biosynthetically

active tissues, with a high demand for glycolytic precursors. This is also supported by

data from lipid rich seeds. PFP is implicated in the inability of wrinkled1 mutant

Arabidopsis seeds to accumulate triacylglycerol – seeds that also accumulate up to five-

times more sucrose than the wild type seeds (Focks and Benning 1998). In addition, the

total lipid content of tobacco seeds constitutively over expressing G. lamblia PFP

increased significantly and the onset of lipid deposition was advanced by up to 48 hours

in the developing transgenic embryos (Wood et al. 2002a, 2002b).

Finally, sugarcane varieties also cycle a significant amount of carbon between the

triose-phosphate and hexose-phosphate pools, for which PFP is at least partially

responsible (Whittaker 1997, Bindon and Botha 2002). The extent of this cycling is also

inversely correlated to sucrose accumulation across varieties (Whittaker 1997) and

maturing internodal tissue (Bindon and Botha 2002). Similarly, in sucrose storing carrot

suspension cells, high respiratory activity stimulates triose-phosphate:hexose-phosphate

cycling, but reduces the cells’ ability to accumulate sucrose (Krook et al. 2000).

Reduced PFP activity should therefore not only directly decrease glycolytic carbon flow

but also reduce the extent of this seemingly wasteful cycle.

Hypothesis 2: PFP influences sucrose metabolism indirectly through its impact on PPi

levels. (a) Its ability to synthesise PPi could contribute to sucrose mobilisation via SuSy.

(b) Its inability to utilise PPi could favour the activity of the H+-translocating vacuolar

pyrophosphatase (VPPase) and in doing so, improve the secondary translocation of

sucrose into the vacuole.

PPi is primarily located in the cytosol at concentrations between 200 and 300 µM,

which are very accurately maintained (Weiner et al. 1987, Takeshige and Tazawa

1989). Moreover, PPi levels in the cytosol are remarkably insensitive to abiotic stresses

such as anoxia or Pi starvation or following the addition of respiratory poisons, which

- 22 -

elicit a significant reduction in cellular ATP pools (Plaxton 1996, Stitt 1998). Cellular

ATP levels on the other hand changes dramatically under these conditions. In addition,

over expressing a soluble alkaline pyrophosphatase from E. coli in transgenic tobacco

and potato plants resulted in plants containing significantly less PPi, which showed a

dramatic phenotype with altered levels of metabolites in primary metabolism and major

changes in their carbohydrate levels, sink-source relations, development and growth rate

(Jellito et al. 1992, Sonnewald 1992). PPi therefore seems to play an essential role in

plant metabolism, growth and development.

A potential role for PFP in sucrose mobilisation through the SuSy and subsequent UDP-

glucose pyrophosphorylase (UGPase, EC 2.7.7.9) catalysed reactions has been proposed

by various authors (Huber and Akazawa 1986, Black et al. 1987, ap Rees 1988). In fact,

the main difference in the PFP-catalysed reaction between sucrose and starch storing

tissues was suggested to be the direction of the net flux, i.e. PPi generation or

consumption respectively, to allow the mobilisation of sucrose in sucrose storing tissues

(Hajirezaei and Stitt 1991). However, this contrasts with the findings of Wong et al.

(1988, 1990) in carrot root and tomato fruits, also sucrose storing tissues, which

suggests that the kinetic characteristics of PFP might be adapted to favour the

gluconeogenic reaction to supply the necessary precursors for sucrose synthesis.

In sugarcane SuSy activity is associated with the elongation of the internodes (Lingle

and Smith 1991) and in general decreases with maturation in internodal tissue (Lingle

and Smith 1991, Zhu et al. 1997, Lingle 1999, Schäfer et al. 2004a) and suspension

cells (Wendler et al. 1990, Goldner et al. 1991). Variation in the ratio between the

breakdown and synthetic activity of SuSy prevents the direct correlation of activity and

sucrose utilisation (Goldner et al. 1991, Schäfer et al. 2004a) and although sucrose

accumulation seems to correspond with a decrease in SuSy activity in suspension cells

(Wendler et al. 1990, Goldner et al. 1991) this does not correspond to similar changes in

PFP activity and the PPi concentrations (Wendler et al. 1990). Additional support that

the PFP reaction and the mobilisation of sucrose via SuSy are not directly linked comes

from transgenic potato plants that over express a soluble pyrophosphatase. Although

- 23 -

sucrose cleavage was inhibited due to PPi deficiency it did not alter the activity of PFP

in these plants (Mustroph et al. 2005).

Although the mobilisation of sucrose via SuSy, using the PPi generated by PFP cannot

be excluded, there is no evidence suggesting that this play an important role in the

accumulation of sucrose in sugarcane. Moreover, even in the case of the relatively

straight forward correlation between cell wall synthesis and the mobilisation of the

required carbon via SuSy activity (Lingle and Smith 1991, Amor et al. 1995), PPi

should not play a major role because the UGPase reaction is not directly involved. In

conclusion, although the inverse correlation between PFP activity and sucrose

concentrations (Whittaker and Botha 1999) apparently fits with the potential of high

PFP activities to lower sucrose concentrations (mobilise sucrose) it is not supported by

the available PFP and PPi data.

Regarding the second part of the hypothesis: Although the sugar concentrations are

probably similar in the apoplast, cytoplasm and vacuole (Welbaum and Meinzer 1990,

Preisser et al. 1992) the vacuolar compartment represents more than 90% of the

intracellular space in mature sugarcane parenchyma cells and is therefore the most

significant sub-cellular compartment where sucrose is stored (Komor 1994). It also

represents a relatively stable compartment for stored sucrose from which very little is

remobilised (Bindon and Botha 2001). Increasing the flux of sucrose into this

compartment therefore has the potential to increase the amount of stored sucrose.

Despite numerous attempts to characterise a potential H+-sucrose antiport system in the

sugarcane tonoplast similar to that of sugar beet (Briskin et al. 1985, Getz and Klein

1995) success has not yet been achieved. Although ATP stimulates sucrose transport

across the tonoplast of sugarcane cells, the mechanism for this is not clear because an

H+-sucrose antiport system could not be unequivocally demonstrated (Williams et al.

1990, Getz et al. 1991). Similarly, although both ATP and PPi can stimulate H+

translocation across the tonoplast, these experimental systems could not yield any

evidence for proton-coupled sucrose translocation either (Williams et al. 1990, Preisser

- 24 -

and Komor 1991). These conflicting results are at least in part due to experimental

difficulties in preparing pure and intact tonoplast preparations from sugarcane cells and

the existence of an H+-sucrose antiport system could therefore not be excluded (see

Moore 1995 for a review). The rest of the discussion will therefore be based on the

assumption that there is indeed an H+-sucrose antiporter in the tonoplast of sugarcane

parenchyma cells that facilitates the active transport of sucrose into the vacuole.

Both vacuolar pyrophosphatase (VPPase, EC 3.6.1.1) and H+-translocating vacuolar

ATPase (VATPase; EC 3.6.1.3) catalyse the electrogenic translocation of protons from

the cytosol to the vacuolar lumen to generate an inside-acidic pH and a cytosol-negative

electrical potential difference, which can be used to drive the secondary transport of

various solutes, including ions, amino acids and sugars, into the vacuole (Sze 1985;

Hedrich and Schroeder 1989; Hedrich et al. 1989). VPPase could therefore theoretically

utilise the phosphoanhydride energy bond in PPi to pump H+ into the vacuole and

thereby activate the secondary transport of sucrose into the vacuole.

In the absence of a soluble inorganic pyrophosphatase, PPi levels in the cytosol can only

be regulated by a combination of the activities of the three PPi utilising enzymes

present, i.e. UGPase, VPPase and PFP. If these three enzymes work collectively to

regulate PPi concentrations it is reasonable to argue that if one of these activities is low /

reduced, one or both of the other two activities will have to increase to reach and

maintain the desired PPi levels - an apparently crucial metabolic parameter as discussed

above. Moreover, because high PPi concentrations will inhibit many biosynthetic

reactions the removal of the excess PPi from the system is crucial to maintain normal

growth and development. This should be especially true when there is a greater need for

the down-regulation of PPi concentrations, e.g. at times of high biosynthetic activity in

young, metabolically active tissues where PPi is a by-product of many biosynthetic

reactions. Inherently low PFP activity could therefore translate into increased VPPase

activity, which should lead to the more efficient energisation of the tonoplast, which in

turn could improve the secondary transport of sucrose into the vacuole. A direct link

- 25 -

between PPi, PFP activity and proton transport across maize tonoplasts was

demonstrated by Dos Santos et al. (2003).

To conclude, although PFP activity is developmentally regulated, the maximum activity

and the ratio between PFP and PFK activity are influenced more by genotype than by

these developmental changes and fine regulation (Whittaker and Botha 1999, Krook et

al. 2000). In a sugarcane genotype with inherently low PFP activity a bigger burden

could therefore rest on VPPase to regulate PPi concentrations and in doing so indirectly

increase the efficiency of all H+-antiport systems, including a possible H+-sucrose

antiport system. This hypothesis clearly relies on the presence of a H+-sucrose antiport

system and should be further investigated.

CONCLUSION

If the negative correlation between PFP expression and sucrose levels in sugarcane is

real, two probable mechanisms through which PFP could impact on sucrose content can

be offered based on the current literature. The first is based on a reduction in total

respiratory flux, resulting in an increased allocation of carbon to sucrose synthesis and

storage. The second is based on the interconnection between cytosolic carbon and PPi

metabolism where the inability of PFP to regulate PPi concentrations could lead to

increased VPPase activity, resulting in the energisation of the tonoplast and improved

translocation of sucrose into the vacuole. However, before these can be evaluated it is

important to establish if there is indeed a direct correlation between PFP activity and

sucrose content. The only direct way of testing this is to alter PFP levels in the same

genetic background through genetic engineering. The primary aim of this study is

therefore to confirm the potential role of PFP in sucrose accumulation in sugarcane,

which could serve as basis for further investigations and genetic manipulation strategies.

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- 33 -

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- 36 -

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- - 37

CHAPTER 3

Purification and characterisation of pyrophosphate: fructose-6-phosphate 1-

phosphotransferase from sugarcane∗

ABSTRACT

Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in sugarcane

internodal tissue is inversely correlated to sucrose content. To help elucidate this

apparent role in sucrose accumulation we determined sugarcane PFP’s molecular and

kinetic properties. Sugarcane PFP was purified 285-fold to a final specific activity of

4.23 µmol min-1mg-1 protein. It contained two polypeptides of 63.2 and 58.0 kDa

respectively, at near equal amounts that cross-reacted with potato PFP-α and –β

antiserum. In gel filtration analyses the native enzyme eluted in three peaks of 129, 245

and 511 kDa, corresponding to dimeric, tetrameric and octameric forms respectively.

Fructose 2,6-bisphosphate (Fru 2,6-P2) influenced the aggregation state of the enzyme.

Both the glycolytic (forward) and gluconeogenic (reverse) reactions had relative broad

pH optima between pH 6.7 and 8.0 and Fru 2,6-P2 shifted the pH optimum for the

forward reaction to a more acidic pH (6.7-7.0). The Fru 2,6-P2 saturation curves were

sigmoidal with approximate Ka values of 69 and 82 nM for the forward and reverse

reactions respectively. The enzyme showed hyperbolic saturation curves for all its

substrates with Km values comparable to that of other plant PFPs, i.e. 150, 37, 39 and

460 µM for fructose 6-phosphate, inorganic pyrophosphate, fructose 1,6-bisphosphate

and inorganic phosphate respectively. This study showed that sugarcane PFP’s

molecular and kinetic characteristics do not differ significantly from that of other plant

PFPs.

KEY WORDS

PFP, Pyrophosphate: fructose-6-phosphate 1-phosphotransferase, sucrose, sugarcane

∗ To be submitted to Journal of Plant Physiology

- - 38

INTRODUCTION

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) is found

exclusively in the cytosol and is one of three enzymes catalysing the interconversion of

fructose 6-phosphate (Fru 6-P) and fructose 1,6-bisphosphate (Fru 1,6-P2), a key

reaction in primary carbohydrate metabolism (see Plaxton 1996 for a review). High PFP

activity has been associated with strong sink strength, i.e. sucrose cleavage (ap Rees et

al. 1985, Black et al. 1987, Xu et al. 1989, Hajirezaei and Stitt 1991), increased

glycolytic flux (Dennis and Greyson 1987, Hatzfeld et al. 1989, Mertens et al. 1991),

gluconeogenic flux (Botha and Botha 1991a, Botha and Botha 1993) and regulation of

cytosolic PPi concentrations (Simcox et al. 1979, ap Rees et al. 1985, Dancer and ap

Rees 1989, Claassen et al. 1991) but no clear physiological function has yet emerged

(Stitt 1989, Trípodi and Podestá 1997, Murley et al. 1998).

In most plants PFP is present as a heterotetramer consisting of two catalytic (PFP-β)

and two regulatory (PFP-α) subunits, approximately 66 and 60 kDa respectively (Yan

and Tao 1984, Kruger and Dennis 1987, Wong et al. 1990, Botha and Botha 1991a,

Nielsen 1994). Depending on various cellular conditions the relative amount and ratio

between the α- and β-subunits may also vary, influencing the regulatory properties of

the enzyme (Kruger and Dennis 1987, Botha and Botha 1991a, 1991b, Theodorou et al.

1992, Theodorou and Plaxton 1994). The reaction catalysed by PFP is thought to be

close to equilibrium in vivo (Edwards and ap Rees 1986), its activity is strongly

activated by Fru 2,6-P2 (Van Schaftingen et al. 1982, Kombrink et al. 1984) and Fru

1,6-P2 (Sabularse and Anderson 1981, Nielsen 1995) and inhibited by Pi (Kombrink and

Kruger 1984, Botha et al. 1986, Stitt 1989) and PPi (Stitt 1989). In addition, its activity

can be modulated by pH (Yan and Tao 1984, Dancer and ap Rees 1989, Trípodi and

Podestá 1997) and its molecular composition (Kruger and Dennis 1987, Botha and

Botha 1991a, 1991b), suggesting it to be an adaptive enzyme, which should respond

sensitively to developmental and environmental cues (Edwards and ap Rees 1986) as is

also suggested by the environmental sensitivity of its activator Fru 2,6-P2 (Van Praag et

al. 1997).

- - 39

Despite all these characteristics, no significant changes in the visible phenotype or

metabolic fluxes and only small changes in metabolite concentrations are observed in

transgenic potato and tobacco plants in which PFP activity is reduced up to 97%

(Hajirezaei et al. 1994, Paul et al. 1995, Nielsen and Stitt 2001). This is true even when

these plants are exposed to limiting phosphate and nitrogen conditions and cold stress.

Similarly, transgenic tobacco plants over expressing a non-regulated PFP from Giardia

lamblia also do not show dramatic phenotypic or physiological effects (Wood et al.

2002a, 2002b). The alteration induced only a small decrease in starch accumulation in

both source and sink tissues and an increase in the total lipid content of the seeds. In

addition, the onset of lipid deposition was advanced by up to 48 hours in the developing

transgenic embryos (Wood et al. 2002b).

In sugarcane PFP activity in internodal tissue is inversely correlated to sucrose content

across commercial varieties and a segregating F1 population (Whittaker and Botha

1999). Differences in activity are reflected by corresponding changes in the relative

amount of the PFP-β subunit, pointing to coarse regulation. These results are consistent

with the general consensus that PFP catalyses a net glycolytic flux (see Stitt 1990 for

review) and also with the view that PFP synthesises PPi which is needed for the

mobilisation of sucrose through sucrose synthase (SuSy, EC 2.4.1.13; Huber and

Akazawa 1986, Black et al. 1987, ap Rees 1988). It contrasts, however, with the

findings of Wong et al. (1988, 1990) who suggested that the kinetic characteristics of

PFP in sucrose storing tissues, e.g. carrot root and tomato fruits, might be adapted to

favour the gluconeogenic reaction, thereby maintaining substrate levels for sucrose

synthesis. In addition, findings in in vitro grown potato tubers suggest a sugar-inducible

effect on coarse control of PFP (Appeldoorn et al. 1999).

The contradiction between the strong inverse correlation of PFP activity and sucrose

content in sugarcane and its apparent redundancy as suggested by transgenic studies

could be due to possible unique properties of the enzyme in this species. The aim of the

current work was therefore to investigate the expression, molecular and kinetic

characteristics of sugarcane PFP to determine if it has any features that are in accord

- - 40

with its apparent role in the sucrose storage phenotype. Here we report that most of

sugarcane PFP’s properties are similar to that of other plant PFPs although some notable

differences do occur. The possible role of these differences in sucrose accumulation is

also discussed.

MATERIALS AND METHODS

Chemicals

All chemicals, auxiliary enzymes, cofactors, substrates and kits were of molecular

biology grade and were obtained from Sigma-Aldrich Fine Chemicals (St. Louis,

Missouri, USA), Roche Diagnostics (Mannheim, Germany) or Promega (Madison,

Wisconsin, USA). Antiserum for potato PFP-α and -β were obtained from Dr NJ

Kruger (Oxford, England) and has previously been described (Kruger and Dennis

1987). The anti-rabbit IgG-alkaline phosphatase conjugate was obtained from Roche.

Plant material and sample preparation

Plant tissues

Mature, non-flowering, field grown (Stellenbosch, South Africa) sugarcane plants,

Saccharum spp. hybrid varieties NCo310 and N19, were randomly selected and

harvested. The leaf with the uppermost visible dewlap, the node it was attached to and

internode immediately above it was defined as leaf-, node- and internode one

respectively, according to the system of Kuijper (Van Dillewijn, 1952). Internodes

selected for analysis were excised from the stalk and ground to a fine powder in liquid

nitrogen after the rind was carefully removed. Leaf roll samples were ground in a

similar manner and consisted of the first 50 mm of tissue just above the apical

meristem, only including leaves younger than leaf -1 (minus one).

Callus

Callus growth was induced in the dark from leaf roll explants on MS nutrient media

containing 3 mg l-1 2,4-D (Taylor et al. 1992, Snyman et al. 1996). Type 2 yellowish,

mucilaginous callus and type 3 white embryogenic callus, consisting of densely

- - 41

cytoplasmic cells (Taylor et al. 1992), were harvested after four to five weeks and

ground in liquid nitrogen. All prepared tissue samples were stored in sealed plastic

containers at –80oC until used. A combination of type 2 and type 3 calli was harvested

and used for the large-scale purification of PFP.

Enzyme extraction and purification

Crude extracts

Proteins were extracted from approximately 500 mg of the ground tissues by stirring it

for 15 min on ice in 2.5 volumes of freshly prepared extraction buffer (100 mM Tris

(pH 7.2), 5 mM MgCl2, 150 mM KCl, 5 mM EDTA, 5% (m/v) PEG 6,000, 0.05% (v/v)

IGEPAL, 2% (m/v) PVPP, 10 mM DTT and 1x Complete™ protease inhibitor cocktail

(Roche)). Cell debris was precipitated by centrifugation (15 min at 10,000 g) to render a

clear supernatant in which enzyme activity was determined.

PFP Purification

All the extraction and purification steps were performed on ice or at 4oC. Thirty grams

of ground tissue was extracted with 2.5 volumes of extraction buffer (as for crude

extracts) by stirring it for 20 min. The homogenate was clarified through centrifugation

(15 min at 10,000 g) and subsequent filtration through nylon mesh. PEG 6,000 was

slowly added to the filtrate to a final concentration of 15%, stirred for 15 min and

incubated statically for a further 15 min. The fractionated proteins were precipitated as

above and resuspended in 10 ml phosphocellulose buffer (100 mM PIPES (pH 6.6), 2

mM MgCl2, 3 mM EDTA, 10% (v/v) glycerol and 5 mM DTT). The equivalent of 0.5 g

dry weight, washed and pre-equilibrated phosphocellulose (washed with 10 volumes

(m/v) 1 M KOH (pH>10) for 10 min, rinsed with water until the pH was neutral,

washed with 10 volumes (m/v) 10% HCl (pH<3) for 10 min, rinsed again with water as

above and pre-equilibrated three times with five bed volumes of phosphocellulose

buffer for 15 min each time) was added to the solution and stirred for 30 min. This

slurry was applied to a 30 mm column and washed until the A280 decreased to the

baseline. Bound proteins were eluted with a 5, 10, 20 mM Na2PPi (in phosphocellulose

buffer) step gradient and collected in 5 ml fractions. Fractions containing more than

20% of the total activity were pooled and one volume of ion exchange buffer (20 mM

- - 42

HEPES (pH 7.5), 2 mM MgCl2, 1 mM EDTA, 10% (v/v) glycerol and 5 mM DTT) was

added to this. The protein was adsorbed at 1 ml min-1 onto an anion-exchange column

(5 ml HiTrap™ Q-Sepharose, Amersham Biosciences, Uppsala, Sweden) and washed

with ion exchange buffer until the A280 decreased to the baseline. PFP was eluted with

60 ml of a linear 0 to 500 mM KCl gradient in ion exchange buffer; collecting 2 ml

fractions. Fractions containing more than 25% of the total activity were pooled, frozen

in liquid N2 and stored at -80oC.

SDS-PAGE and protein blotting

One microgram of purified protein and 20 µg of crude protein extracts were resolved on

a 10% (m/v) SDS-PAGE according to the modified method of Moorhead and Plaxton

(1991) as described by Whittaker (1997). For the silver staining of gels Amersham

Biosciences’ PlusOneTM silver staining kit for proteins (Cat. Nr. 17-1150-01) was used

according to the manufacturer’s instructions. Spot densitometry was done using the

AlphaEaseFC software (version 4.0.1) of Alpha Innotech Corp. (San Leandro, CA,

USA). Protein blots were done according to the methods described by Whittaker and

Botha (1999). In brief, after the peptides were transferred to nitrocellulose membranes

the blots were blocked overnight in 4% (m/v) BSA and then incubated in a 1:500

dilution of the specific antiserum. Thereafter the blots were washed and incubated with

a 1:2,000 dilution of a goat anti-rabbit IgG-alkaline phosphatase conjugate (Roche),

washed again and developed through the enzymatic cleavage of 5-bromo-4-chloro-3-

indolyl-phosphate, using nitroblue tetrazolium as a stain enhancer (Blake et al. 1984).

Gel filtration chromatography

Gel filtration chromatography was done at room temperature on a Superose® 6 HR

10/30 column (Amersham Biosciences) using a ÄKTAprime FPLC-system (Amersham

Biosciences). Thirty milli-units of PFP was applied to the column and fractionated in

gel filtration buffer (100 mM Tris (pH 7.2), 5 mM MgCl2, 150 mM KCl, 5 mM EDTA,

10 mM DTT and ½x Complete™ protease inhibitor cocktail (Roche)) at a flow rate of

0.3 ml min-1. One hundred microliter fractions were collected and assayed for PFP

- - 43

activity. PFP’s native molecular weight was determined by linear regression analysis

using ferritin (440 kDa), aldolase (158 kDa), BSA (67 kDa) and ovalbumin (43 kDa) as

molecular weight markers. To determine the influence of Fru 2,6-P2 on the aggregation

state of the enzyme it was incubated for 1h in the presence of 25 µM Fru 2,6-P2 and the

sample was fractionated in the presence of 2 µM Fru 2,6-P2 (in the above buffer).

PFP assays and kinetic analysis

PFP activity was measured using the conditions as described by Whittaker and Botha

(1999). All enzyme assays were carried out in a final volume of 250 µl at 30oC. Activity

was measured by quantifying the oxidation of NADH (forward reaction) or the

reduction of NADP+ (reverse reaction) at 340nm using a Power WaveX microplate

scanning spectrophotometer (Bio-Tek Instruments, Winooski, Vermont, USA). Kinetic

constants were determined by varying the different substrate concentrations and the

analysis of the data by non-linear regression analyses (two-parameter rectangular

hyperbola) using the Sigma Plot 2000 (V6.1), Enzyme Kinetics Module (V1.0) software

package (SPSS, Chicago, Illinois, USA).

RESULTS

Purification and molecular characterization

PFP activity in different sugarcane tissues

PFP activity varies significantly between various sugarcane tissue types; activity was

therefore measured in crude extracts of several tissue types from two commercial

sugarcane varieties, NCo310 and N19, to determine the best source for PFP protein. For

the two tissue types originating directly from plants, PFP activity (U g-1 fresh weight)

was 12 to 18 times higher in the leaf roll than in internode seven. The specific activity

(U mg-1 protein) of the leaf roll extract was also 2.5 to 3.5 times higher than that of the

internode seven extract (Fig. 1). Compared to the leaf roll tissue, type 2 and 3 callus

yielded up to five times more PFP activity per gram fresh weight, at a four times higher

specific activity (Fig. 1). There was no significant difference in yield between the two

callus types, but in general N19 callus yielded PFP at twice the specific activity of

- - 44

NCo310. Compared to the plant tissues, callus also had the additional advantage of very

low levels of phenolic contamination, which increased the overall efficiency of the

extraction.

Fig. 1 PFP activity in various sugarcane tissue types as determined in crude extracts. Protein extracts were prepared from the two sugarcane varieties, NCo310 (310) and N19, and the tissue types sampled were internode seven (I7), leaf roll (LR), type 2 (C2) and type 3 (C3) callus.

Subsequently PFP was partially purified (PEG-fractionated) from both internode seven

and callus tissue of variety N19 to determine if the enzymes present in these tissues had

similar kinetic properties. Based on the very big differences in the reported kinetic

parameters for plant PFPs (refer to Table 2 in Chapter 2), it is evident from the data

presented in Table 1 that the kinetic parameters for the forward reaction of sugarcane

PFP extracted from internodal and callus tissue respectively are comparable.

Table 1. Kinetic parameters for the forward reaction of partially purified sugarcane PFP from internodal and callus tissue.

Internode 7 Callus Parameter

0µM Fru 2,6-P2 50µM Fru 2,6-P2 0µM Fru 2,6-P2 50µM Fru 2,6-P2

Km (Fru 6-P) (mM) 0.690 ± 0.042 0.207 ± 0.019 0.594 ± 0.020 0.120 ± 0.013

Km (PPi) (mM) 0.056 ± 0.004 0.035 ± 0.003 0.061 ± 0.005 0.043 ± 0.004

Vmax (U mg-1 protein) 0.007 ± 0.001 0.054 ± 0.006 0.359 ± 0.054 1.045 ± 0.071

0

0.05

0.1

0.15

0.2

0.25

310-I7

310-LR

N19-I7

N19-LR

310-C2

310-C3

N19-C2

N19-C3

PFP

acti

vity

(U m

g-1 p

rote

in)

- - 45

Purification

Based on these findings it was decided to purify sugarcane PFP from N19 type 2 and 3

calli. The results for a typical purification are presented in Table 2. The enzyme was

purified 285-fold to a final specific activity of 4.28 and 3.18 U mg-1 protein for the

forward and reverse reactions respectively, with an overall recovery of 14%. The

inclusion of a protease inhibitor cocktail (Complete™, Roche) was essential; without

these inhibitors up to 65% of the activity was lost during the first purification step. PFP

activity eluted as a single peak during both the phosphocellulose and Q-Sepharose

chromatography steps. The final preparation, which was used for all kinetic analyses,

was essentially free (less than 1% of PFP activity) of contaminating aldolase,

hexosephosphate isomerase, phosphatase and pyrophosphatase activity.

Table 2. Purification of PFP from sugarcane callus.

Step Activity a

(U)

Yield

(%)

Protein

(mg)

Specific activity

(U mg-1)

Purification

(fold)

Crude extract 9.00 100.0 335.92 0.015 1.0

5-15% PEG 7.38 82.0 175.52 0.046 3.1

Phosphocellulose b 2.13 23.7 5.63 0.361 24.1

Anion exchange b 1.25 13.9 1.61 4.281 285.4 a Measured in the forward direction at pH 7.25. b Pooled fractions.

Molecular composition

SDS-PAGE analyses using both silver- and Coomassie staining revealed two dominant

polypeptides at 63.2 ± 3.8 and 58.0 ± 2.3 kDa respectively, corresponding to the two

subunits of PFP found in most other plant species (Fig. 2). Judged by these analyses the

two peptides were present in near equal amounts. Moreover, the average ratio between

the 63.2 and 58.0 kDa peptides was calculated to be 1 : 1.2, using spot densitometry.

- - 46

Fig. 2 SDS-PAGE and immunoblot analysis of purified sugarcane PFP. (1) Silver stained gel with 1µg of the purified protein and immunoblots of the same samples using potato PFP-α (2) and PFP-β (3) antisera respectively.

Protein blot analyses were done using antisera generated against potato PFP-α and -β

subunits respectively (Kruger and Dennis 1987). Although the putative β-subunit cross-

reacted specifically with the potato PFP-β antisera the PFP-α antisera cross-reacted to

both the peptides (Fig 2). The PFP-α antisera was also less immunoreactive than the

PFP-β antisera.

Molecular weight

The native size of the PFP enzyme was determined using gel filtration chromatography.

PFP activity eluted in three peaks with apparent molecular masses of 524 ± 18, 239 ± 8

and 132 ± 4 kDa respectively (Fig. 3). In the absence of Fru 2,6-P2 the 132 kDa peak

dominated (96% of total activity) but in the presence of Fru 2,6-P2 more than 65% of

the total activity was eluted in the 524 kDa peak. The 239 kDa peak represented

approximately 3% of the total activity in the absence of Fru 2,6-P2 and only 1% in the

presence of Fru 2,6-P2 .

1 2 3

66 kDa

97 kDa

45 kDa

1 2 3

66 kDa

97 kDa

45 kDa

- - 47

Fig. 3 Gel filtration chromatography of purified sugarcane PFP in the absence ( ) and presence ( ) of Fru 2,6-P2. Arrows indicate the elution volumes of ferritin (a), aldolase (b), albumin (c) and ovalbumin (d) respectively.

Kinetic and regulatory characterisation

pH dependence

The pH dependence of the activity was similar for the forward and reverse reactions.

Both reactions had relatively broad activity ranges, retaining more than 80% of their

maximum activity between pH 6.75 and 8.0 (Fig. 4). The pH optima for both reactions

were between 7.25 and 7.5. In the absence of Fru 2,6-P2 the pH optimum for the

forward reaction shifted slightly to a more acidic pH (6.75-7.0). The pH activity profile

of the reverse reaction in the absence of Fru 2,6-P2 had a very broad activity range (6.0-

9.0). Compared to a HEPES-acetate based buffer, a Tris-HCl based buffer did not

inhibit PFP activity as was reported for potato, mung bean and Shamouti orange PFP

(Agosti et al. 1992, Van Praag et al. 2000). All subsequent kinetic studies for both the

forward and reverse reactions were performed at pH 7.25.

Substrate saturation kinetics and Fru 2,6-P2 activation

In general, the enzyme showed hyperbolic saturation curves for all the substrates. The

kinetic parameters of the enzyme were determined using non-linear regression analyses.

Apparent Km and Vmax values obtained for the various substrates of PFP are summarised

in Table 3. The enzyme’s affinity for Fru 6-P, PPi and Fru 1,6-P2 was increased 27-,

2.5- and 16-fold respectively by the addition of 50 µM Fru 2,6-P2.

0.0

20.0

40.0

60.0

80.0

100.0

10.0 12.0 14.0 16.0 18.0 20.0 22.0Ve (ml)

Rel

ativ

e PF

P ac

tivity

a cb d

0.0

20.0

40.0

60.0

80.0

100.0

10.0 12.0 14.0 16.0 18.0 20.0 22.0Ve (ml)

Rel

ativ

e PF

P ac

tivity

aa ccbb dd

- - 48

Fig. 4 pH dependence of sugarcane PFP activity. PFP activity in the presence (open symbols) and absence (closed symbols) of Fru 2,6-P2 in the forward (A) and reverse direction (B). A MES based buffer ( and ) was used at the low pH values and a Tris based buffer ( and ) at high pH values. The potential inhibitory effect of Tris was tested with a HEPES based buffer ( and ).

In contrast, Fru 2,6-P2 decreased the enzyme’s affinity for Pi almost 2-fold (Table 3). In

the presence of 50 µM Fru 2,6-P2 and saturating substrate concentrations Vmax was

stimulated 2-fold in the forward direction and 1.5-fold in the reverse direction (Table 3).

At subsaturating concentrations of Fru 6-P (5 mM) and Fru 1,6-P2 (0.5 mM) Vmax

increased 5- and 3-fold for the forward and reverse reactions respectively, in the

presence of 50 µM Fru 2,6-P2 (Fig. 2). The Fru 2,6-P2 saturation curves were sigmoidal

with approximate Ka values of 69.3 ± 10.1 and 82.2 ± 14.7 nM for the forward and

reverse reactions respectively at the above mentioned substrate concentrations.

Table 3. Kinetic and regulatory properties of sugarcane PFP at saturating substrate concentrations.

Fru 2,6-P2 concentration Parameter

0µM 50µM

Forward reaction

Kmapp (Fru 6-P) (mM) 4.03 ± 0.29 0.15 ± 0.02

Kmapp (PPi) (mM) 0.090 ± 0.006 0.037 ± 0.004

Vmax (U mg-1 protein) 2.13 ± 0.06 4.28 ± 0.10

Reverse reaction

Kmapp (Fru 1,6-P2) (mM) 0.62 ± 0.10 0.039 ± 0.005

Kmapp (Pi) (mM) 0.27 ± 0.04 0.46 ± 0.06

Vmax (U mg-1 protein) 2.26 ± 0.14 3.26 ± 0.08

0.00.51.01.52.02.53.03.54.0

5.0 6.0 7.0 8.0 9.0 10.0

pH value

PFP

activ

ity (U

mg-1

pro

tein

)

A.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

5.0 6.0 7.0 8.0 9.0 10.0

pH value

PFP

activ

ity (U

mg-1

pro

tein

)

B.

0.00.51.01.52.02.53.03.54.0

5.0 6.0 7.0 8.0 9.0 10.0

pH value

PFP

activ

ity (U

mg-1

pro

tein

)

A.

0.00.51.01.52.02.53.03.54.0

5.0 6.0 7.0 8.0 9.0 10.0

pH value

PFP

activ

ity (U

mg-1

pro

tein

)

A.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

5.0 6.0 7.0 8.0 9.0 10.0

pH value

PFP

activ

ity (U

mg-1

pro

tein

)

B.

0.0

0.2

0.4

0.6

0.8

1.0

1.2

5.0 6.0 7.0 8.0 9.0 10.0

pH value

PFP

activ

ity (U

mg-1

pro

tein

)

B.

- - 49

DISCUSSION

PFP activity in different sugarcane tissues

Initial investigations identified type 2 and 3 callus as an ideal source for sugarcane PFP.

Callus did not only yield up to 75 times more PFP per gram fresh weight than internodal

tissue, but also had a 20 times higher specific activity and very little contaminating

phenolics. Comparative molecular and kinetic studies on partially purified PFP

extracted from internodal and callus tissues confirmed that the enzymes had similar

properties. In combination with earlier findings that only a single allele of the PFP-β

gene is expressed in various sugarcane tissues (Huckett et al. 2001) it is reasonable to

argue that PFP isolated from callus will have similar molecular and kinetic properties

than the PFP from internodal tissues.

The specific activity of PFP in the crude extracts from internode seven tissue was

comparable to that obtained in previous studies (Lingle and Smith 1991, Whittaker and

Botha 1999). Similarly, the specific activity in the NCo310 callus was the same as that

obtained by Wendler and co-workers (1990) in sugarcane suspension cultures, although

N19 callus yielded PFP at approximately twice that specific activity. In contrast to the

difference in specific activity between the two varieties, the variation between the two

different callus types was not significant. This suggests that PFP activity is genetically,

rather than developmentally determined in sugarcane callus cultures, similar to that

reported for different lines and cell types in carrot suspension cultures (Krook et al.

2000). Finally, the specific activities of PFP in the various sugarcane tissues reported

here, falls comfortably within the range published for other plant tissues, including

sugar storing tissues such as carrot tap root (0.012 U mg-1 protein, Wong et al. 1988),

tomato fruits (0.15 U mg-1 protein, Wong et al. 1990) and ripe banana fruits (0.012 U

mg-1 protein, Turner and Plaxton 2003).

Purification and molecular properties

Sugarcane PFP was purified 285-fold to a final specific activity of 4.23 and 3.18 U mg-1

protein for the forward and reverse reactions respectively. Fru 2,6-P2 had a profound

effect on the apparent molecular weight of the native enzyme. In the absence of Fru 2,6-

- - 50

P2 96% of the total activity eluted in a 132 kDa peak corresponding to the dimeric form

of the enzyme. Under these conditions the apparent tetrameric and octameric forms of

the enzyme represented only 3% and 1% of the total activity respectively. The addition

of Fru 2,6-P2 induced aggregation, which resulted in the direct conversion of the

dimeric form into the octameric form. Similar influences have been described for pea

(Wu et al. 1984), spinach (Balogh et al. 1984) and carrot (Wong et al. 1988) PFP, but in

these cases the aggregations were always sequential, i.e. from the dimeric to the

tetrameric (Balogh et al. 1984, Wu et al. 1984) or from the tetrameric to the octameric

state (Wong et al. 1988). Based on this data the kinetic parameters for the enzyme

reported above, in the absence or presence of Fru 2,6-P2, should therefore be for the

dimeric and octameric forms respectively. Moreover, PFP should be predominantly in

the octameric form in vivo, based on the calculated concentrations of Fru 2,6-P2 in

sugarcane (Lingle and Smith 1991, Whittaker and Botha 1999).

SDS-PAGE analyses revealed that the purified enzyme consisted of two polypeptides at

near stoichiometric amounts, corresponding to an α- (63 kDa) and β-subunit (58 kDa).

This data matched the sequence deduced peptide sizes, which became available more

recently, i.e. 67 and 61 kDa for the α- and β-subunits respectively (unpublished data,

refer to Telles et al. 2001). The putative β-subunit interacted specifically with potato

PFP-β antisera, confirming the antigenic similarity between these two peptides.

Alignment of their amino acid sequences lent further support to their functional

complementarity indicating 81% homology based on amino acid identity. In contrast the

potato PFP-α antiserum apparently cross-reacted with both peptides. Any cross-

reactivity will be surprising because both the PFP-α and -β antisera are highly specific

for their respective target peptides in potato (Kruger and Dennis 1987). Moreover, there

is only a 41% homology between the deduced amino acid sequences of the sugarcane

PFP-α and -β proteins and an even lower homology (38%) between potato PFP-α and

sugarcane PFP-β. A more feasible explanation might therefore be that the PFP-α

peptide was subjected to post-translational or in vitro modification that resulted in a

second peptide similar in size to the PFP-β protein. The apparent lower

immunoreactivity of the PFP-α antisera could be explained by the lower homology

- - 51

between the sugarcane and potato proteins; 65% compared to the 81% for PFP-β.

Alternatively it might be an artefact based on the dispersal of the signal if the PFP-α

peptide is indeed subjected proteolytic activity.

Kinetic and regulatory properties

Both reactions had relatively broad pH optima between pH 6.75 and 8.00 and the

reverse reaction was particularly insensitive to pH in the absence of Fru 2,6-P2. In

addition, Fru 2,6-P2 shifted the pH optimum of the forward reaction to a slightly more

basic pH, i.e. from 6.75 to 7.5, and the biggest degree of activation was also measured

at the higher pH values (7.25 – 7.5). A pH response to Fru 2,6-P2 activation, although

exactly the opposite, has only been reported for a few PFPs. In rice seedlings (Enomoto

et al. 1992), Pi stressed black mustard suspension cells (Theodorou and Plaxton 1996)

and pineapple leaves (Trípodi and Podestá 1997) Fru 2,6-P2 shifts the enzyme’s pH

optimum to a more acidic pH and the degree of activation increases in this direction. All

these authors argued that this activation pattern suggests a glycolytic role for PFP under

conditions that result in a decrease in cytosolic pH, e.g. anoxia and night acidification in

CAM plants. The significance of this slight change in optimum pH is not clear,

especially in light of the fact that maximum activation, for both the forward and reverse

reactions, takes place at the optimum pH for the activated reactions.

The Fru 2,6-P2 saturation curves for the forward and reverse reactions were sigmoidal

with approximate Ka values of 0.069 and 0.082 µM respectively. Although the reported

Fru 2,6-P2 levels in sugarcane (~3µM; Lingle and Smith 1991, Whittaker and Botha

1999) is much higher than the Ka values reported here the in vivo activation of PFP will

depend much on the prevailing concentrations of Pi and other phosphorylated

intermediates (Kombrink and Kruger 1984, Theodorou and Kruger 2001). Fru 2,6-P2

induced a relatively small increase in the Vmax (2x) in the forward direction and a

similar increase (1.5x) in the reverse direction. The fact that sugarcane PFP’s Vmax is

influenced very similarly by Fru 2,6-P2 in both directions is dissimilar to most other

plant PFPs for which the forward reaction’s Vmax is in general increased much more

(>5x) than the reverse (Kombrink et al. 1984, Wong et al. 1988, Enomoto et al. 1992,

- - 52

Nielsen 1994, Theodorou and Plaxton 1996, Trípodi and Podestá 1997 and Turner and

Plaxton 2003) or where the reverse reaction is not affected by Fru 2,6-P2 at all (Wong et

al. 1990, Van Praag et al. 2000).

The enzyme showed hyperbolic saturation curves for all the substrates with Km values

comparable to that of other plant PFPs. In contrast to the findings in other sugar storing

tissues, i.e. carrot (Wong et al. 1988) and tomato PFP (Wong et al. 1990), Fru 2,6-P2

significantly increased (27x) the enzymes’ affinity for Fru 6-P. The suggestion of these

authors that PFPs in sugar storing tissues might favour gluconeogenic flux because of

their low affinity for Fru 6-P especially under conditions that favour high Fru 2,6-P2

levels, therefore does not hold true for sugarcane PFP. Based on the reported levels for

the relevant metabolites in sugarcane internode seven tissue, Fru 6-P, 85µM; PPi,

189µM; Fru 1,6-P2, 37µM; and Pi, 5.1mM (Whittaker and Botha 1997), PFP could

operate close to its Vmax in vivo. In conjunction with the calculated substrate ratios that

suggest that the PFP-catalysed reaction is close to equilibrium (Whittaker and Botha

1997) and the similarity in the activation patterns of both the forward and reverse

reactions reported above, it suggests a very responsive system that could react in a

flexible manner to changes in metabolite levels.

Implications to the sucrose accumulation phenotype

Sugarcane PFP’s molecular and kinetic properties do not differ significantly from that

of other plant PFPs in a way that clearly suggests a direct role for it in the accumulation

of sucrose, e.g. reduced glycolytic or increased gluconeogenic carbon flux. The

information presented here therefore supports the study by Whittaker and Botha (1999)

in that the apparent link between PFP activity and sucrose accumulation is based on the

total amount of catalytic activity present in the tissue and not the fine regulation thereof.

It is important to stress that the above mentioned study correlates PFP activity with

sucrose content specifically in internode seven. Although the sucrose content of older

internodes is significantly higher (Welbaum and Meinzer 1990, Botha et al. 1996)

- - 53

internode seven has one of the highest sucrose accumulation rates (Whittaker and Botha

1997, 1999). The relevance of this lies in the fact that the influence of PFP in sucrose

concentration might be of a transient nature, i.e. might be obscured over time. Internode

seven represents tissues with high metabolic activity and carbohydrate flux (Whittaker

and Botha 1997, Bindon and Botha 2002). A perturbation in the metabolic network

could therefore lead to an altered flux, e.g. into sucrose accumulation, which could

temporarily lead to altered concentrations, but which would be restored over time to

basal levels. This argument is supported by the transient influence of reduced PFP

activity on sucrose concentrations in transgenic potato tubers. Although the final

concentration is unchanged the flux into sucrose in the growing (metabolically active)

tubers is 13-times higher in comparison to the wild type (Hajirezaei et al. 1994). The

argument is used to support the hypothesis that PFP influence flux rather than the ability

of the tissue to store sucrose. It is therefore probably more accurate to refer to PFP’s

influence on the sucrose accumulation rate in these tissues instead of their sucrose

content.

Lower PFP activity could improve sucrose accumulation directly by (i) increasing the

concentration of precursors for sucrose synthesis, i.e. reduced glycolytic flux (Wong et

al. 1988, Focks and Benning 1998, Krook et al. 2000) or indirectly by (ii) limiting the

production of PPi which could be used for the mobilisation and utilisation of sucrose

through SuSy (ap Rees et al. 1985, Black et al. 1987, Huber and Akazawa 1986, Xu et

al. 1989, Hajirezaei and Stitt 1991). However, the role of PFP in sucrose accumulation

in sugarcane can only be irrefutably demonstrated in transgenic plants with altered PFP

activity, which is currently under investigation in our laboratories.

Acknowledgements

The South African Sugar Association, the South African Department of Trade and

Industry and Stellenbosch University sponsored this work. We thank Dr NJ Kruger

(Department of Plant Sciences, Oxford, England) for his kind gift of potato PFP-α and -

β antisera.

- - 54

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Whittaker A. Pyrophosphate dependent phosphofructokinase (PFP) activity and other aspects of sucrose metabolism in sugarcane internodal tissues. PhD thesis, University of Natal, South Africa. 1997; pp 1-187.

Whittaker A and Botha, FC. Carbon partitioning during sucrose accumulation in sugarcane internodal tissue. Plant Physiology 1997; 115: 1651-1659.

Whittaker A and Botha, FC. Pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase activity patterns in relation to sucrose storage across sugarcane varieties. Physiologia Plantarum 1999; 107: 379-386.

Wong JH, Kang, T, and Buchanan, BB. A novel pyrophosphate fructose-6-phosphate 1-phosphotransferase from carrot roots. Relation to PFK from the same source. FEBS Lett 1988; 238: 405-410.

Wong JH, Kiss, F, Wu, M-X, and Buchanan, BB. Pyrophosphate Fructose 6-P 1-Phosphotransferase from tomato fruit. Plant Physiol 1990; 94: 499-506.

Wood SM, King, SP, Kuzma, MM, Blakeley, SD, Newcomb, W, and Dennis, DT. Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase overexpression in transgenic tobacco: physiological and biochemical analysis of source and sink tissues. Can J Bot 2002a; 80: 983-992.

Wood SM, Newcomb, W, and Dennis, DT. Overexpression of the glycolytic enzyme Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) in developing transgenic tobacco seeds results in alterationsin the onset and extent of storage lipid deposition. Can J Bot 2002b; 80: 993-1001.

Wu M-X, Smyth, DA, and Black, CC. Regulation of pea seed pyrophosphate-dependent phosphofructokinase: Evidence for interconversion of two molecular forms as a glycolytic regulatory mechanism. Proc Natl Acad Sci USA 1984; 81: 5051-5055.

Xu D-P, Sung, S-JS, Loboda, T, Kormanik, PP, and Black, CC. Characterization of sucrolysis via the uridine diphosphate and pyrophosphate-dependent sucrose synthase pathway. Plant Physiol 1989; 90: 635-642.

Yan T-F and Tao, M. Multiple forms of pyrophosphate: D-fructose-6-phosphate 1-phosphotransferase from wheat seedlings. Regulation by fructose-2,6-bisphosphate. J Biol Chem 1984; 259: 5087-5092.

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CHAPTER 4

Development and characterisation of transgenic systems for the manipulation of

PFP activity in sugarcane∗

ABSTRACT

To manipulate PFP activity in sugarcane the endogenous PFP-β coding sequence and

two monogenic, non-plant PFP genes, those of G. lamblia and P. freudenreichii, were

cloned and characterised. The 1668 bp sugarcane PFP-β cDNA sequence showed high

homology to other plant PFP-β sequences and was subsequently used to construct

antisense and co-suppression expression vectors for the down-regulation of PFP

activity. The activity and kinetic parameters of the non-plant PFPs were determined

using purified proteins produced in bacterial expression systems. Based on these

parameters it was concluded that the non-plant PFPs should function at least at ½Vmax in

transgenic sugarcane plants. Polyclonal antibodies raised against these two enzymes

showed no cross reactivity with sugarcane PFP. The two non-plant genes were used to

construct plant expression vectors for the up-regulation of PFP activity, either

constitutively or specifically in the phloem.

KEY WORDS

PFP, Pyrophosphate: fructose-6-phosphate 1-phosphotransferase, bacterial expression,

expression vector, transgenic sugarcane, sucrose metabolism

INTRODUCTION

Pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) is most

probably present in all plants (Stitt 1990) and in a limited number of prokaryotes and

lower eukaryotes such as Propionibacterium shermanii (O’Brien et al. 1975),

∗ Parts of this work have been published as follows: 1) Patent PA128025/ZA, A high level, stable, constitutive promoter element for plants. 2) Groenewald et al. 2000. Plant Cell Rep 19: 1098-1101.

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Giardia lamblia (Mertens 1990), Entamoeba histolytica (Reeves et al. 1974),

Rhodospirillum (Pfleiderer and Klemme 1980) and Euglena gracilis (Miyatake et al.

1984). The enzyme catalyses the reversible conversion of fructose 6-phosphate (Fru 6-

P) and pyrophosphate (PPi) to fructose 1,6-bisphosphate (Fru 1,6-P2) and inorganic

phosphate (Pi) (Reeves et al.1974). This is thought to be a near-equilibrium reaction in

vivo and would therefore be able to respond to cellular metabolism in a very flexible

manner (Edwards and ap Rees 1986).

Although the exact physiological role of PFP in plants is still unknown, its kinetic

properties have been studied in some detail and it is evident that it plays an important

role in sucrose cycling, breakdown and subsequent respiratory carbon flow (Sabularse

and Andersen 1981, Kombrink et al. 1984, Bertagnolli et al. 1986, Botha and Small

1987, Stitt 1990). PFP activity in sugarcane internodal tissues is inversely correlated to

sucrose content across commercial varieties and a segregating F1 population (Whittaker

and Botha 1999). These differences in activity are also reflected by corresponding

changes in the relative amount of the PFP-β subunit, implying coarse regulation.

Potentially, the levels of both PPi and Fru 6-P could control the rate of glycolysis, i.e.

respiratory flux (Plaxton 1996 and references therein) and, in addition, PFP has been

implicated in the determination of sink strength (Edwards and ap Rees 1986, Botha et al.

1992, Black et al. 1995). Manipulation of PFP activity in sugarcane could therefore have

an impact on carbon distribution between stored sucrose and respiration.

Plant PFPs are usually heterotetramers of approximately 250 kDa, composed of two α-

and two β-subunits with molecular weights of approximately 66 and 60 kDa

respectively (Kruger and Dennis 1987). The β-subunits are the catalytic subunits while

the α-subunits are involved in the regulation of enzyme activity through fructose 2,6-

bisphosphate (Fru 2,6-P2) (Yan and Tao 1984, Carlisle et al. 1990, Cheng and Tao

1990, Van Praag 1997). In contrast, the PFPs from the prokaryotes and lower

eukaryotes are homodimers, which are insensitive to Fru 2,6-P2 activation (O’Brien et

al. 1975, Mertens 1990).

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PFP activity has been increased indirectly in transgenic plants by over expressing Fru

2,6-P2 (Kruger and Scott 1994, Scott and Kruger 1995), or directly through the

expression of a heterologous enzyme (Wood et al. 2002a and b). The digenic nature of

plant PFP and the fact that its activity is finely regulated in vivo complicates the direct

up-regulation of activity in transgenic plants. However, both these difficulties can be

overcome by the use of homodimeric PFP enzymes such as those isolated from

prokaryotes and lower eukaryotes. The successful expression of any of these PFP genes

in transgenic plants should be reflected in an increase in the in vivo PFP activity,

because only a single gene product is necessary to produce a functional enzyme and the

resulting enzyme is not sensitive to the endogenous regulatory mechanisms in plants.

Down-regulation of PFP activity, on the other hand, can be done by down regulating the

expression of any one of the two endogenous genes encoding each of the two subunits

of plant PFP. Using homology-dependent gene silencing technology, gene expression

can be specifically down-regulated by transforming the plant with either an antisense or

untranslatable form of the target gene (Fagard and Vaucheret 2000 and references

therein).

Here we report the establishment of various transformation systems that will allow the

up- and down-regulation of PFP activity in sugarcane. The goals for this work did not

only include the cloning of the relevant gene sequences and the construction of

appropriate plant expression vectors but also the expression, purification and

characterisation of the heterologous PFP proteins to confirm their bio-activity and

potential properties under in vivo conditions. Finally, antisera were raised against the

two purified proteins to enable the easy characterisation of transgenic plants.


Chemicals and enzymes

All chemicals, enzymes, including their reaction buffers, cofactors and kits were of

molecular biology grade and were obtained from the sources mentioned in Chapter 3.

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Recombinant DNA technology

Cloning and expression vectors

All PCR derived fragments were cloned directly into the pGEM®-T Easy cloning

vector (Promega) and the pGEX-4T1 bacterial expression system (Pharmacia,

Milwaukee, USA; GENBANK U13853) was used for the expression and purification of

recombinant proteins. For constitutive gene expression in transgenic plants the plant

expression vector pUBI 510 (ECACC deposit reference number: 00042603,

Groenewald et al. 2000) was used. Gene expression in this vector is driven by the

CaMV 35S (Odell et al. 1985) and the maize polyubiquitin (UBI-1, Christensen et al.

1992) promoters in tandem and, in addition, also contains the first intron of UBI-1,

which acts as an enhancer. Vectors for the phloem specific expression of transgenes

were constructed by using the rolC promoter (Chilton et al. 1982, Groenewald 1999)

from the plasmid pBINrolC (provided by Dr. J. Schell, Max Planck Institute, Köln,

Germany). Standard cloning techniques were used during the construction of these

vectors and characterisation of the final constructs was done by restriction analyses and

sequencing (Sambrook et al. 1989).

RT-PCR

RT-PCR was done using the TitanTM RT-PCR system (Roche) according to the

manufacturer’s specifications.

DNA sequencing

Automated DNA sequencing was performed with an Applied Biosystem ABI Prism 373

Genetic Analyser using an ABI BigDyeTM terminator cycle sequencing ready reaction

kit according to the manufacturer's recommendations (Perkin-Elmer, Boston,

Massachusetts, USA).

Library construction and screening

mRNA was isolated from the leaf roll of sugarcane commercial variety N19 (Bugos et

al.1995) and cDNA was synthesised using the Universal RiboClone® cDNA Synthesis

System (Promega) according to the manufacturer’s instructions. A cDNA library was

constructed using Stratagene’s (La Jolla, California, USA) Lambda ZAP II system

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according to the manufacturer’s instructions. A library with a titre of 3 x 109 plaque

forming units per millilitre (pfu ml-1) was obtained. Library screening was done

according to standard methods as described earlier (Groenewald 1999).

P. freudenreichii cultivation and isolation of gDNA

P. freudenreichii was grown for five days at 37oC under anaerobic conditions in a

medium containing 0.5% (m/v) yeast extract, 0.2% (m/v) Peptone P, 36.7 mM KH2PO4,

1.0% (v/v) lactic acid, 0.1% (v/v) Tween 80 and 0.001% (m/v) Hemin. To isolate

gDNA the cells were harvested through centrifugation, resuspended in lysis buffer (50

mM Tris (pH 8.0), 1 mM EDTA, 0.2% (m/v) lysozyme) and incubated at 37oC for 30

min. Thereafter SDS and proteinase K was added to final concentrations of 1% (m/v)

and 50 µg ml-1 respectively and the suspension was incubated at 50oC for 4 h. The

suspension was extracted twice with an equal volume of chloroform:isoamyl alcohol

(Chl:IAA, 24:1) and the aqueous phase was transferred to a small glass beaker and

overlaid with 2.5 volumes of –20oC, 96% (v/v) ethanol. High molecular weight gDNA

was spooled out with a glass hook and resuspended in 1 ml TE buffer (10 mM Tris, 1

mM EDTA, pH 8.0). The gDNA was treated with RNase (0.2 mg ml-1) for 30 min at

37°C and the Chl:IAA (24:1) extraction was repeated. Finally, the gDNA was

quantified spectrophotometrically.

Expression, purification and characterisation of recombinant proteins

Bacterial expression system and purification of proteins

To verify the bioactivity and kinetic properties of the homodimeric PFP gene products

and to purify the proteins for the generation of polyclonal antibodies the two genes were

cloned into the pGEX-4T1 bacterial expression system (Pharmacia). This system

expresses the recombinant protein as a GST-fusion to allow the easy purification of the

product. Both the production, preparation of crude extracts and affinity purification of

the recombinant proteins were done according to the instructions of the manufacturer.

G. lamblia PFP cultures were grown at 37oC while the P. freudenreichii cultures had to

be grown at 22oC to ensure the production of a soluble product. The GST-fusion

proteins were purified using Glutathion Sepharose 4B affinity chromatography as

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recommended by the manufacturer. Proteins used for the kinetic characterisation of the

enzymes were eluted after Thrombin digestion to remove the GST-peptide and the

GST-fusion proteins used to raise antibodies were eluted using reduced glutathion.

Polyclonal antiserum

Rabbits were immunised by injecting them with eight fractions of 100 µg purified

protein according to the method described by Bellstedt et al. (1987). Whole blood, from

which the antiserum was obtained, was collected after 38 days.

Enzyme assays and kinetics

PFP activity was measured using the conditions as described by Whittaker (1997) with

the exception that Fru 2,6-P2 was omitted from the reaction mixture. All enzyme assays

were carried out in a final volume of 250 µl at 30oC. Activity was measured by

quantifying the oxidation of NADH (forward reaction) or the reduction of NADP+

(reverse reaction) at 340 nm using a Power WaveX microplate scanning

spectrophotometer (Bio-Tek Instruments, Winooski, Vermont, USA). Kinetic constants

were determined by varying the different substrate concentrations and the analysis of

the data by non-linear regression analyses (two parameter rectangular hyperbola) using

the Sigma Plot 2000 (V6.1), Enzyme Kinetics Module (V1.0) software package (SPSS,

Chicago, Illinois, USA).

SDS PAGE and protein blot analyses

SDS PAGE and protein blot analyses were done as described in Chapter 3.

RESULTS AND DISCUSSION

Selection of non-plant PFPs

The primary structure of a transgene can determine the efficiency with which it is

expressed in transgenic plants (Perlak et al. 1991, Sutton et al. 1992). Potential non-

plant PFPs, to be used for the up-regulation of PFP activity in transgenic sugarcane,

were therefore evaluated in terms of the availability of their gene sequences and

differences in their nucleotide and amino acid sequences, GC-content and codon usage

(Table 1). Based on these criteria two genes were identified. G. lamblia PFP consists of

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two 59.8 kDa monomers, which are encoded by a 1635 bp gene (Rozario et al. 1995),

while the 43.3 kDa monomer of P. freudenreichii PFP is encoded by a 1215 bp gene

(Ladror et al. 1991). The most important properties of these two genes are summarised

and compared to plant PFPs in Table 1.

Table 1. Comparison of plant and non-plant PFPs.

Origin Protein Gene Exons %GC GENBANK Activator Reference

Castor bean 250 kDa heterotetramer α- 1.9 kb β- 1.7 kb

α- 19 β- 16

α- 44.4% β- 44.5%

α- Z32849 β- Z32850 Fru 2,6-P2

Todd et al. 1995

Potato 250 kDa heterotetramer α- 1.9 kb β- 1.5 kb

α- n.a. β- n.a.

α- 43.4% β- 44.6%

α- M55190 β- M55191 Fru 2,6-P2

Kruger and Dennis 1987

G. lamblia 120 kDa homodimer 1.6 kb None 50.3 % U12337 None Rozario et al. 1995

P. freudenreichii 90 kDa homodimer 1.2 kb None 66.7 % M67447 None Ladror et al. 1991

n.a. = not available

Isolation and characterisation of the PFP gene sequences

Sugarcane PFP-β gene

To ensure optimal antisense and co-suppression mediated gene silencing in transgenic

sugarcane the endogenous PFP-β gene sequence was isolated. A set of degenerate

primers was designed (PFP-B4 and PFP-B2, Table 2), based on the consensus sequence

of the castor bean and potato PFP-β gene sequences available in the international

database; GENBANK accession numbers Z32850 and M55191 respectively

(www.ncbi.nlm.nih.gov). These primers were used to amplify a 248 bp cDNA fragment,

spanning exons eight; nine and ten, from sugarcane leaf roll RNA using the RT-PCR

technique. Subsequent cloning and characterisation of the fragment confirmed its

identity as being the PFP-β gene.

Table 2. Primers used to amplify a 248 bp fragment of sugarcane PFP-β.

Primer Binding site Sequence

PFP-B4 bp 802 – 821 (forward) 5’- CAC ATT ACI TTI GIA TGC GC –3’

PFP-B2 bp 1049 – 1029 (reverse) 5’- TCA TCI ACA ACA TCA TGI GCC –3’

- 67 -

The amplified PFP-β cDNA fragment was used as a probe to screen a sugarcane leaf

roll cDNA library for putative PFP-β clones. Isolated clones were sequenced to verify

the inserts’ identity and integrity. One such clone, PFP#5, contained a 1135 bp

fragment, which represents 53% (875/1668 bp) of the PFP-β coding sequence, spanning

exons 8 to 16 (Fig. 1).

ATGGCGGCGC CGAGCGGACC ATCACCTGGG ACTGGGAGGT TGGCGTCGGT TTACAGCGAG GTGCAGACGA GCCGCCTCCA TCACGCGATC CGGCTCCCCT CCGTCCTCTG CTCCCAATTC TCCCTCGTCG ATGGACCTCC CAGCTCAGCC ACGGGGAACC CGGATGAGAT CGCGAAGCTG TTCCCTAACT TGTTTGGGCA GCCGTCGGCG ACATTGGTGC CGGCCAAAGA GGCGGTGGAG GGGAAGGCGC TGAAGGTCGG GGTGGTGCTC TCTGGTGGAC AAGCACCCGG TGGGCACAAT GTGATCTGCG GTATCTTCGA TTTCTTGCAG AAACACGCAA AGGGAAGCAC AATGTATGGA TTCAAAGGAG GCCCAGCAGG GGTGATGAAG TGCAAGTACG TCAAACTCAA TACCGATTTC GTCTATCCCT ACAGAAACCA GGGTGGTTTT GATATGATCT GTAGTGGAAG GGATAAGATT GAAACACCAG AGCAGTTTAA GCAAGCCGAA GATACAGCCA ACAAACTTGA GTTGGACGGA CTTGTTGTTA TTGGACGGGA CGATTCAAAT ACTCATGCTT GCCTCTTTGC TGAATACTTC AGGAGTAAAA ATTTGAAAAC CCGTGTCATT GGCTGCCCAA AGACCATTGA TGGTGATCTC AAATGCAAAG AGGTTCCAAC CAGTTTTGGA TTTGACACTG CATGCAAGAT CTATTCAGAA ATGATTGGAA ATGTCATGAT TGATGCCCGA TCAACTGGAA AATATTATCA CTTTGTACGG CTTATGGGGC GTGCTGCTTC TCACATTACA TTGGGATGCG CTTTGCAAAC ACACCCCAAT GCTGCACTCA TTGGGGAAGA GGTTGCTGCA AAGAAGCAAA CCCTTAAGAA CGTCACAAAC TACATTACTG ATATCATCTG CGAGCGTGCA GATCTTGGTT ACAACTATGG TGTTATCCTT ATACCAGAAG GCCTGATTGA TTTCATCCCA GAGGTGCAGA ATATCATTGC TGAATTGAAT GAAATTTTGG CACATGATGT TGTTGATGAG GCAGGGGCCT GGAAAAGCAA GCTTCAGCCT GAATCAAAGG AGCTGTTTGA GTTTTTGCCC AAAACTATTC AGGAGCAACT TATGCTTGAA AGGGGCCCCC ATGGCAATGT TCAGGTTGCA AAAATTGAAA CCGAGAAAAT GCTTATTAGC ATGGTGGAAA CTGAACTGGA GAAGAGAAAA GCAGAGGGGA GATACTCTGC ACATTTCAGA GGGCAAGCTC ATTTCTTTGG GTACGAAGGA AGATGTGGCC TTCCTACCAA TTTTGATTCT AACTATTGCT ATGCATTAGG CTATGGGGCT GGTGCCCTTC TCCAAAGTGG GAAGACAGGA CTTATTTCAT CGGTTGGCAA CCTTGCGGCT CCAGTAGAAG AATGGACTGT TGGTGGAACA GCATTGACAT CACTGATGGA TGTGGAGAGG AGGCATGGCA AGTTCAAGCC AGTGATCGAG AAGGCTATGG TGGAACTTGA TGCTGCACCT TTCAAGAAAT ATGCATCAAT GCGGGATGAG TGGGCCACCA AGAACAGATA CATCAGCCCT GGCCCCATCC AGTTCAGTGG CCCTGGAAGT GATGACTCGA ACCACACTTT GATGCTGGAA CTCGGTGCTG AGTTATAGAG ATGCGTCCTT TGCTTATTTT TGTTTCTTAC AGTTTTGGGA GTGGAGACTG GACACTGGGT CTCCTGGAGC AGCCTGCAGT CTCCATATTG TGAATTGTTT AATAAGAGGT TCGATGTGAG TTTTCTGCGT AGCGGACTGG ATGTAGCAAA TAAGAACTGG TTTTAGCATT TTTTGTATGA TTTACGCACC AACTGACTTG TCTTGTAACC CTGATTCTGT TCCACTGGTT GCAATCTCGT GAGAATGAAC AAGTTGATAT GAGGCTAAAT CGGAATTCCT GCAGCCCGGG GGATCCACTA GTTCTAGAGC GGCCGCCACC GCGGTGGAGC TCCAGCTTTT TTCCCTTTAG TCAGGGTAAT TCGAACTGCG A

Fig. 1 Coding sequence and 3’ UTR of the sugarcane PFP-β gene. The initiation and termination codons are indicated in bold and the sequence of the cDNA clone, PFP#5, is underlined.

The rest of the insert consisted of a 260 bp 3’ untranslated sequence. Comparing the

sequence to the international database (BLASTN software; www.ncbi.nlm.nih.gov/cgi-

bin/BLAST) confirmed its identity as being PFP-β - it was 74.9% and 75.0%

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homologous to the castor bean and potato PFP-β sequences respectively. Moreover, this

sequence was also 96.9% homologous to a recently published maize sequence

(GENBANK AY104192). This gene fragment was therefore used in the construction of

antisense and co-suppression plant expression vectors, aimed at down-regulating the

expression of the sugarcane PFP-β gene. Subsequently, the complete sugarcane PFP-β

coding sequence, as presented in Fig. 1, was cloned and characterised.

Non-plant PFP genes

The G. lamblia PFP gene, originally characterised by Rozario et al. (1995, GENBANK

U12337), was obtained from Dr D Dennis (Performance Plants, Queens University,

Kingston, Canada). To isolate the P. freudenreichii PFP gene specific primers were

designed to amplify the complete coding sequence from gDNA. The primer sequences

were based on the published P. freudenreichii PFP gene sequence of Ladror et al.

(1991, GENBANK M67447) and BamH I restriction sites were introduced into the

primers to facilitate the subsequent cloning of the gene (Table 3). As expected, a 1.2 kb

fragment was amplified from P. freudenreichii gDNA, which was cloned into the

pGEM®-T Easy cloning vector and sequenced. Comparison with the international

database confirmed the cloned sequence to be the PFP gene of P. freudenreichii

(BLASTN software; www.ncbi.nlm.nih.gov/cgi-bin/BLAST).

Table 3. Primer sequences used to amplify the P. freudenreichii PFP gene from gDNA. The initiation and termination codons are indicated in bold and the introduced BamH I sites are underlined.

Primer Binding site Sequence

Prop-PFP-F bp -11 – 15 (forward) 5’-GG CCC GGA TCC ATG GTG AAA AAG GTC–3’

Prop-PFP-R bp 1240 – 1213 (reverse) 5’-TC GCC GGA TCC CTA TTA CGC GGC GGC G–3’

Expression, purification and kinetic characterisation of non-plant PFPs

Because the non-plant PFP genes were earmarked for the over expression of PFP

activity in transgenic sugarcane, it was imperative to verify the bioactivity of the two

gene products and to confirm their potential effectiveness in transgenic sugarcane

plants, i.e. determine their kinetic properties under sugarcane’s physiological

- 69 -

conditions. Both genes were therefore cloned into the pGEX-4T1 bacterial expression

system, which would express the enzymes as GST-fusion proteins to facilitate the

purification of the recombinant enzymes.

Although both systems expressed active enzymes, P. freudenreichii PFP was only

detectable when the cultures were grown at low temperatures, e.g. 22°C (Fig. 2).

Subsequent protein blot analyses indicated that this was not due to incorrect folding of

the peptides but rather the complete absence of soluble protein (results not shown).

Preparative cultures were prepared based on these findings and the recombinant

enzymes were purified using affinity chromatography and subsequent thrombin

cleavage to remove the GST-fusion. G. lamblia and P. freudenreichii PFP were

respectively purified to final specific activities of 3.82 and 2.53 U mg-1 protein.

Fig. 2 Activity of recombinant G. lamblia and P. freudenreichii PFP in crude extracts from bacterial expression systems. BL 21 cells represent the untransformed E.coli strain used, pGEX-4T1 is the cloning vector without an insert, p95.624 is the vector containing the G. lamblia PFP gene and pBPP-4T1 contains the P. freudenreichii PFP gene.

Both enzymes had relatively broad pH optima, between 6.5 and 7.8, for both the

forward and reverse reactions (Fig. 3) and displayed typical Michaelis-Menten kinetics

for all four substrates. The kinetic parameters of the enzymes were determined with

non-linear regression analyses as described in the materials and methods section. The

kinetic parameters determined for the two enzymes are summarised and compared to

the concentrations of these substrates in sugarcane in Table 4. In addition, both enzymes

were also shown to be completely active in the absence of Fru 2,6-P2 (Fig. 4).

0.0

0.1

0.2

0.3

0.4

BL 21cells

pGEX-4T1

p95.624(37°C)

pBPP4T1(37°C)

pBPP4T1(22°C)

PFP

activ

ity (U

mg-1

prot

ein)

0.0

0.1

0.2

0.3

0.4

BL 21cells

pGEX-4T1

p95.624(37°C)

pBPP4T1(37°C)

pBPP4T1(22°C)

0.0

0.1

0.2

0.3

0.4

BL 21cells

pGEX-4T1

p95.624(37°C)

pBPP4T1(37°C)

pBPP4T1(22°C)

PFP

activ

ity (U

mg-1

prot

ein)

- 70 -

pH value

Rel

ativ

e ac

tivity

0

20

40

60

80

100

4 5 6 7 8 9 100

20

40

60

80

100

4 5 6 7 8 9 10pH value

Rel

ativ

e ac

tivity

A. B.

pH value

Rel

ativ

e ac

tivity

0

20

40

60

80

100

4 5 6 7 8 9 100

20

40

60

80

100

4 5 6 7 8 9 10pH value

Rel

ativ

e ac

tivity

A. B.

Fig. 3 pH dependence of G. lamblia( ) and P. freudenreichii ( ) PFP activity for the forward (A) and reverse (B) reactions respectively.

Table 4. Kinetic properties of G. lamblia and P. freudenreichii PFP compared to the metabolite concentrations in sugarcane culm (Whittaker and Botha 1999).

Km (µM) Metabolite

G. lamblia P. freudenreichii Concentration in sugarcane

culm (µM) Fru 6-P 79.60 196.70 85-178 PPi 37.57 41.40 166-290 Fru 1,6-P2 26.98 37.83 32-46 Pi 1331.89 1491.83 3400-6100 pH optimum 6.5 - 7.8 6.5 – 7.8 6.7 – 8.0

Fig. 4 Influence of Fru 2,6-P2 on the activity of G. lamblia (open bars) and P. freudenreichii (filled bars) PFP, compared to that of a plant (potato, ) PFP. Vmax for G. lamblia, P. freudenreichii and potato PFP was 3.54, 2.63 and 0.61 µmol min-1 mg-1 protein respectively.

Based on the Km values for the four different substrates and their concentrations in

sugarcane, both enzymes should function at least at 1/2Vmax in transgenic sugarcane

Fru 2,6-P2 concentration (µM)

Rel

ativ

e ac

tivity

0

20

40

60

80

100

10-5 10-4 0.5 1 5 10 1005010-3 10-2 10-1

Fru 2,6-P2 concentration (µM)

Rel

ativ

e ac

tivity

0

20

40

60

80

100

10-5 10-4 0.5 1 5 10 1005010-3 10-2 10-10

20

40

60

80

100

10-5 10-4 0.5 1 5 10 1005010-3 10-2 10-1

- 71 -

plants. In addition, the confirmed insensitivity of these enzymes to Fru 2,6-P2 suggest

that they would be insensitive to the usual in vivo regulatory mechanisms of PFP in

sugarcane. The successful expression of the proteins in transgenic plants should

therefore be translated into increased PFP activity.

After the kinetic characterisation of the enzymes additional protein was purified for the

production of polyclonal antibodies to facilitate the future characterisation of putative

transgenic clones. In this instance the whole GST-fusion protein was eluted from the

affinity column using reduced glutathion. Rabbits were immunised against the purified

proteins and 38-day antiserum was tested at a 1:500 dilution to verify the specificity of

the raised antibodies (Fig. 5). The polyclonal antibodies showed no cross reactivity to

sugarcane PFP, which makes it ideal for the characterisation of transgenic plants. This

lack of cross reactivity is not surprising considering the low homology between these

peptides on amino acid level, i.e. the highest percentage homology (identity) between

any of the four peptides is the 47% between G. lamblia PFP and the sugarcane PFP-β

protein.

Fig. 5 Protein blot analysis using thirty-eight day serum raised against the GST fusions of G. lamblia (A) and P. freudenreichii PFP (B). The following protein extracts were loaded in the respective lanes: 1) purified G. lamblia PFP, 2) purified P. freudenreichii PFP, 3) untransformed E. coli, 4) pGEX-4T1 transformed cells, 5) crude extract of the GST-G. lamblia PFP fusion protein, 6) crude extract of the GST-P. freudenreichii PFP fusion protein, 7) crude extract of sugarcane internode seven protein (variety NCo 310).

97

6645

30

20

97

66

45

30

20

kDa 1 2 3 4 5 6 7 kDaA. B.

1 2 3 4 5 6 7

97

6645

30

20

97

66

45

30

20

kDa 1 2 3 4 5 6 7 kDaA. B.

1 2 3 4 5 6 7

- 72 -

Construction of plant expression vectors

Six plant expression vectors were constructed for the genetic manipulation of PFP

activity in sugarcane. These included vectors for the up- and down-regulation of PFP

activity, either constitutively or specifically in the phloem. A tandem promoter construct

consisting of the CaMV-35S (Odell et al. 1985) and maize UBI-1 (Christensen et al.

1992) promoters, previously shown to drive high level expression in sugarcane tissues

(Groenewald et al. 2000), was used as constitutive promoter and the rolC promoter

(Chilton et al. 1982, Groenewald 1999) was used to confer phloem specific expression.

The general properties of the vectors are summarised in Table 5. All the expression

vectors were designed for direct transformation protocols, i.e. particle bombardment,

and had to be co-transformed with a vector containing a selectable marker, e.g. NPT II.

Plasmid maps of two of these expression vectors are presented in Fig. 6 as examples.

Table 5. Properties of the plant expression vectors constructed to manipulate PFP expression in transgenic sugarcane.

Vector Promoter Gene Regulation Specificity

pEGP 510 35S-UBI-1 G. lamblia Up Constitutive

pEPP 510 35S-UBI-1 P. freudenreichii Up Constitutive

pEGP 800 rolC G. lamblia Up Phloem

pASPc 510 35S-UBI-1 Sugarcane PFP-β Down Constitutive

pUSPc 510 35S-UBI-1 Sugarcane PFP-β Down Constitutive

pASPc 800 rolC Sugarcane PFP-β Down Phloem

Fig. 6 Schematic maps of two of the plant expression vectors constructed for the genetic manipulation of PFP activity in sugarcane.

pASPc 800 6114 bp

rolC-p

nos-t

Amp

EcoR I 10Hind III 400

Xba I 2833

Xho I 1Hind III 5

SC-PFP-β

BamH I 1121Pst I 1129EcoR I 1139

Pst I 1334

Hind III 2013EcoR V 2170

EcoR I 2309EcoR V 2313Hind III 2322

Xba I 438

pEGP 5107147 bp

35S-pUBI-p

UBI-5’ UTR

UBI-i

CaMV-t

Xba I 1Hind III 400Pst I 405

Sal I 1001

Bgl II 1347

Pst I 2400

BamH I 2410

Xba I 4917Sal I 2405

Amp

EcoR I 1810

G. lamblia PFP

EcoR I 2421EcoR I 4152

Hind III 3462Pst I 3214

Pst I 4004Xba I 4148

A. B.

pASPc 800 6114 bp

rolC-p

nos-t

Amp


Xba I 2833

Xho I 1Hind III 5

SC-PFP-β


Pst I 1334



pASPc 800 6114 bp

rolC-p

nos-t

Amp


Xba I 2833

Xho I 1Hind III 5

SC-PFP-β


Pst I 1334



Xba I 438

pEGP 5107147 bp

35S-pUBI-p

UBI-5’ UTR

UBI-i

CaMV-t


Sal I 1001

Bgl II 1347

Pst I 2400

BamH I 2410


Amp

EcoR I 1810

G. lamblia PFP




Xba I 438

pEGP 5107147 bp

35S-pUBI-p

UBI-5’ UTR

UBI-i

CaMV-t


Sal I 1001

Bgl II 1347

Pst I 2400

BamH I 2410


Amp

EcoR I 1810

G. lamblia PFP




A. B.

- 73 -

CONCLUSION

The main aim of the work described here was to establish relevant transformation

systems for the up- and down-regulation of PFP activity in sugarcane; six plant

expression vectors were constructed which should do so, either constitutively or phloem

specifically. The sugarcane PFP-β gene was cloned and characterised for the first time

and two monogenic, non-plant PFP genes were also cloned and characterised. In

addition the bio-activity of these heterologous PFP proteins, to be used for the up-

regulation of activity, was confirmed and their potential properties in sugarcane under in

vivo conditions were determined. Finally, antisera were raised against the two purified

heterologous PFP proteins, which should facilitate the characterisation of transgenic

plants.

Although the over expression of PFP activity in sugarcane will be investigated to

complement this study the rest of the work described here will focus only on the down-

regulation of PFP activity in transgenic plants to assess the apparent inverse correlation

described earlier.

Acknowledgements

The South African Sugar Association and the South African Department of Trade and

Industry sponsored this work. We thank Dr D Dennis (Performance Plants, Queens

University, Kingston, Canada) for his kind gift of the G. lamblia PFP coding sequence,

Dr J Schell (Max Planck Institute, Köln, Germany) for providing us with the rolC

promoter and Prof D Bellstedt for raising the antiserum.

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- 78 -

CHAPTER 5

Down-regulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase

activity in sugarcane enhances sucrose accumulation in immature internodes∗

ABSTRACT

Pyrophosphate: fructose 6-phosphate 1-phosphotransferase (PFP) activity was

successfully down-regulated in sugarcane using constitutively expressed antisense and

untranslatable forms of the sugarcane PFP-β gene. In young internodal tissue activity

was reduced by to 70% while no residual activity could be detected in mature tissues.

The transgenic plants showed no visible phenotype or significant differences in growth

and development under greenhouse and field conditions. Sucrose concentrations were

significantly increased in the immature internodes of the transgenic plants but not in the

mature internodes. This contributed to an increase in the purity of the immature tissues,

resembling an early ripening phenotype. PFP activity was inversely correlated with

sucrose content in the transgenic lines. Both the immature and mature internodes of the

transgenic plants had significantly higher fibre contents. These findings suggest that

PFP influences the ability of biosynthetically active sugarcane cells to accumulate

sucrose but that the equilibrium of the glycolytic intermediates, including the stored

sucrose, is restored when ATP-dependent phosphofructokinase and the residual PFP

activity is sufficient to sustain the required glycolytic flux as the tissue matures.

Moreover, it suggests a role for PFP in glycolytic carbon flow, which could be rate

limiting under conditions of high metabolic activity.

KEY WORDS

PFP, Pyrophosphate: fructose-6-phosphate 1-phosphotransferase, carbon metabolism,

sucrose, transgenic sugarcane, gene silencing

∗ To be submitted to Transgenic Research. Aspects of this chapter have also been published in: 1) Patent PA128784/ZA, The regulation and manipulation of sucrose content in sugarcane. 2) Groenewald and Botha 2001. Proc. Int. Soc. Sugarcane Technol. 24, 592-594.

- 79 -

INTRODUCTION

Pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) catalyses

the reversible conversion of fructose 6-phosphate (Fru 6-P) and pyrophosphate (PPi) to

fructose 1,6-bisphosphate (Fru 1,6-P2) and inorganic phosphate (Pi); one of the principle

reactions in glycolysis (Reeves et al. 1974, Carnal and Black 1979). In sugarcane, PFP

activity in internodal tissues is inversely correlated with sucrose content and positively

related to total respiration, across commercial varieties and within a segregating F1

population (Whittaker and Botha 1999). In addition, a significant amount of carbon is

cycled between the triose-phosphate and hexose phosphate pools, for which PFP is at

least partially responsible (Bindon and Botha 2002). The extent of this cycling

decreases concomitantly with sucrose accumulation in mature internodal tissue (Bindon

and Botha 2002). Similarly, in sucrose storing carrot cell suspensions a high respiratory

activity stimulates the flow of hexose phosphates towards the respiratory pathway and

the recycling (back-flow) of the triose-phosphates, but reduces the cells’ ability to

accumulate sucrose (Krook et al. 2000). PFP activity is directly linked to these carbon-

partitioning patterns and, as in sugarcane, the ratio between PFP and fructose 6-

phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activity is influenced more by

genotype than by development (Whittaker and Botha 1999, Krook et al. 2000). In

sugarcane, differences in PFP activity also correspond to changes in the amount of the

PFP-β subunit, pointing to coarse regulation (Whittaker and Botha 1999).

Fructose 2,6-bisphosphate (Fru 2,6-P2) is a potent activator of PFP activity (Sabularse

and Anderson 1981, Van Schaftingen et al. 1982). In general it increases the Vmax of

both the forward (glycolytic) and reverse (gluconeogenic) reactions and the enzyme’s

affinity for the substrates (decreases Km) (Sabularse and Andeson 1981, Van

Schaftingen et al. 1982, Botha et al. 1986, Theodorou and Plaxton 1996). In contrast,

the enzymes purified from carrot taproots and ripe tomato fruits have a decreased

affinity for Fru 6-P (increased Km) in the presence of Fru 2,6-P2 (Wong et al. 1988,

1990). This led the authors to speculate that these enzymes might be adapted to favour

the gluconeogenic reaction in sucrose storing tissues. We have, however, recently

shown that this does not hold true for sugarcane PFP, where saturating Fru 2,6-P2

- 80 -

concentrations decreased the Km of Fru 6-P more than 27 times (Chapter 3). In addition,

the inverse correlation between sucrose concentrations and PFP activity in sugarcane

(Whittaker and Botha 1999) is consistent with the general consensus that PFP catalyses

a net glycolytic flux (Stitt 1990 and references therein).

Studies on transgenic plants, in which PFP activity was down-regulated by as much as

99%, gave contradictory evidence for the role of PFP. Transgenic potato (Hajirezaei et

al. 1994) and tobacco (Paul et al. 1995, Nielsen and Stitt 2001) plants show no changes

in visible phenotype and in general the metabolite concentrations and fluxes are not

significantly impacted upon. Although these studies provide evidence that PFP catalyses

a net glycolytic flux, they also suggest that it does not contribute to the control of this

flux (Hajirezaei et al. 1994, Nielsen and Stitt 2001). In addition, PFP was shown to be

responsible for the recycling of the triose-phosphates in non-photosynthetic tissues

(Hajirezaei et al. 1994). These studies also show that plant tissues are able to

compensate for the large decrease in PFP activity by the allosteric activation of the

remaining PFP and by the activation of PFK (Hajirezaei et al. 1994, Paul et al. 1995,

Nielsen and Stitt 2001). Overall, one of the most significant observations that can be

made from these studies is the apparent different response of the various tissues to

decreased PFP activity. Moreover, Fru 2,6-P2 concentrations are significantly increased

in metabolically active tissues, e.g. growing (sink) and sprouting (source) potato tubers

(Hajirezaei et al. 1994) and growing (sink) tobacco leaves, particularly at the base

(Nielsen and Stitt 2001), in contrast to mature tubers and leaves. In addition, although

the final sucrose concentration in mature transgenic tubers is unchanged, the flux into

sucrose in the growing (sink) tubers is 13 times higher when compared to that of the

wild type (Hajirezaei et al. 1994).

Recently an unregulated form of PFP from Giardia lamblia was also expressed in

transgenic tobacco plants, which led to a 20-fold increase in PFP activity (Wood et al.

2002a, 2002b). The total biomass of these transgenic lines is lower and carbon

partitioning is altered in both the source and sink tissues. Although total sucrose

concentrations are unaffected in the leaves (source), starch concentrations are

- 81 -

significantly lower (Wood et al. 2002a). Young seeds (sink) have reduced starch levels

but higher lipid levels, which results in similar total seed carbohydrate concentrations

when compared to the wild type seeds (Wood et al. 2002a). In addition to the increase

in seed lipids the specific composition of fatty acids also changed (Wood et al. 2002b).

Transgenic seeds also display a temporal enhancement in the growth and development

of the young embryo (Wood et al. 2002a).

As suggested for other plant PFPs (Edwards and ap Rees 1986, Weiner et al. 1987,

Sung et al. 1988) the calculated mass-action ration for PFP in sugarcane indicates that

the in vivo reaction is close to equilibrium (Whittaker and Botha, 1997). It therefore

appears that sugarcane PFP might be directly involved in the regulation of carbon-flow

between sucrose synthesis and eventual accumulation on the one hand and the supply of

carbon to respiration and other biosynthetic pathways on the other. To verify this

potential role of PFP in sucrose metabolism in sugarcane and in particular its apparent

role in sucrose accumulation, PFP activity was down-regulated in transgenic plants.

In this paper we report on the effect of the reduction of PFP activity on the sugar yields of

the transgenic sugarcane lines. More than 50% of the lines analysed had a significant

increase in sucrose concentrations in their immature internodes, both in greenhouse and

field grown plants. Although most of the transgenic lines’ mature internodes also gave

higher average sucrose yields these were not significant. Field grown transgenic plants also

had significantly higher fibre content. The inverse correlation between PFP activity and

sucrose content also held true for the transgenic lines.


Chemicals

The general chemicals that were used are described in Chapter 3. Antiserum for potato

PFP-β was obtained from Dr NJ Kruger (Oxford, England) and has previously been

described (Kruger and Dennis 1987). Antisera for G. lamblia and P. freudenreichii PFP

were raised against the recombinant proteins expressed in a bacterial expression system

- 82 -

as described earlier (Chapter 4). The anti-rabbit IgG-alkaline phosphatase conjugate was

obtained from Roche.

Plant material and sample preparation

Non-flowering sugarcane stalks were randomly selected and harvested. Wild type

Saccharum spp. hybrid variety NCo310 plants were used as controls and transgenic

lines were also generated using this variety. Both greenhouse and field grown plants of

various ages were used as described for each experiment. The leaf with the youngest

visible dewlap, the node it was attached to and internode just above it was defined as the

number one (1) leaf, node and internode respectively. For the greenhouse grown plants

the selected internodes were excised from the stalk and ground to a fine powder in

liquid nitrogen after the rind was carefully removed. Prepared samples were kept frozen

and stored at –80oC.

Expression vectors, transformation, selection and regeneration

A partial sequence of the sugarcane PFP-β gene was used to construct both an antisense

and a co-suppression expression vector for the down-regulation of PFP activity in

transgenic sugarcane. In both the constructs the gene sequences were under the control

of a constitutive, tandem CaMV-35S : maize UBI-1 promoter. Commercial sugarcane

variety NCo310 was used to induce callus growth in the dark from leaf roll explants on

MS nutrient media containing 3 mg l-1 2,4-D (Taylor et al. 1992, Snyman et al. 1996).

Type 3 white embryogenic calli, consisting of dense cytoplasmic cells (Taylor et al.

1992), were used as targets for biolistic transformation. Co-transformation was done

with the respective silencing constructs and a similar expression vector containing the

npt-II selectable marker. Selection was done on geneticin-containing media and putative

transgenic plants from independent transformation events were regenerated and

hardened off based on standard protocols (Snyman et al. 1996).

- 83 -

RNA extraction and northern blot analysis

RNA was extracted according to the method of Bugos et al. (1995). Purified RNA was

suspended in diethyl pyrocarbonate (DEPC) treated water and quantified

spectrophotometrically. Fifteen µg of total RNA per sample was loaded on a 1.2% (m/v)

Tris-Borate/EDTA (TBE) prepared agarose gel and developed at 100 V until the bromo

phenol blue dye front migrated eight cm. The gel was trimmed and then equilibrated in

10x SSC-buffer (1.5M NaCl, 0.15M Na3C6H5O7 (pH 6.8)) for 20 min while the

positively charged Nylon membrane was wetted in water before it was equilibrated in

10x SSC-buffer for 10 min. The RNA was transferred overnight to the Nylon membrane

by downward capillary blotting using 10x SSC-buffer at room temperature. After

transfer the RNA was UV cross-linked to the membrane on both sides for 1.5 min at

1200 mJ cm-2. The transgene sequence was used as probe and was radioactively labelled

using 25 µCi [∝-32P] dCTP and a random labelling kit (Amersham). Membranes were

prehybridised in 15 ml RAPIDhybTM hybridization buffer at 65°C for 30 min. The

probe was boiled for 5 min, added to the hybridisation bottle and incubated overnight at

65°C. The hybridised blots were washed twice for 15 min each at 50°C and 55°C in

wash solution 1 (1 x SSC, 0.1% (m/v) SDS) and twice for 15 min each at 60°C and

65°C in wash solution 2 (0.5 x SSC, 0.1% (m/v) SDS). The blots were exposed to a

supersensitive Cyclone Phosphor screen (Packard) for 16 to 18 hours. Hybridisation

was visualised using the CycloneTM Storage Phosphor System (Packard Instrument Co.,

Inc., Meriden, USA).

Protein extraction

Crude protein extracts were prepared from approximately 500 mg of the ground tissues

by stirring it for 15 min on ice in 2.5 volumes of freshly prepared extraction buffer (100

mM Tris (pH 7.2), 5 mM MgCl2, 150 mM KCl, 5 mM EDTA, 5% (m/v) PEG 6000,

0.05% (v/v) IGEPAL, 2% (m/v) PVPP, 10 mM DTT and 1x Complete™ protease

inhibitor cocktail (Roche)). Cell debris was precipitated by centrifugation (15 min at

10000 g) to render a clear supernatant in which enzyme activity was determined.

- 84 -

PFP assays

PFP activity was measured using the conditions as described by Whittaker (1997). All

enzyme assays were carried out in a final volume of 250 µl at 30oC. Activity was

measured by quantifying the oxidation of NADH (forward reaction) or the reduction of

NADP+ (reverse reaction) at 340nm using a Power WaveX microplate scanning

spectrophotometer (Bio-Tek Instruments, Winooski, Vermont, USA).

Sugar extraction and quantification

Soluble sugars were extracted in 70% (v/v) ethanol and 30% HM-buffer (100 mM

HEPES [pH 7.8], 20 mM MgCl2), (1/10, m/v) with incubation at 65ºC overnight. The

samples were then centrifuged at 10,000 x g, for 10 minutes at room temperature. The

supernatant was dried under vacuum and resuspended in 1 ml 10% (v/v) isopropanol.

For sucrose determinations all samples were diluted 10 times. Sucrose, glucose and

fructose concentrations were determined spectrophotometrically using the enzymatic

method described by Bergmeyer and Bernt (1974). Purity was expressed as the

percentage ratio between sucrose and the total sugar pool including glucose, fructose

and sucrose.

Field trial

Nine transgenic lines and a wild type control line were transplanted, using setts, to a

field at the South African Sugarcane Research Institute (SASRI), KwaZulu-Natal, South

Africa. Each line was grown in a single 8 m row, representing eight individual stools

approximately 1 m from each other. Row spacing was 1.5 m. The plant crop was

harvested after approximately 15 months in the winter (July). Three independent

samples of 1.5 to 2 kg each were prepared for each line by the random harvesting of

whole stalks. Excess leaf material and the tops, i.e. the 3-5 youngest internodes of the

cane, were removed as is customary during commercial harvesting. Sample preparation

and analysis was done in a research mill using standard, industry-scale procedures

(Anonymous 1977). Data collected included the total soluble sugar yield (Brix % DW

and Brix % cane, i.e. FW), sucrose yield (Pol % cane), purity (%) and fibre content

- 85 -

(Fibre % cane) as is standard for the sugar industry. After the first harvest the sugarcane

was allowed to ratoon and was again harvested after 16 months during early summer

(November). Although the harvesting was done as before, on this occasion the stalks

were divided approximately in half to yield a top and a bottom half that were analysed

separately. At this age the cane consisted of approximately 34 harvestable internodes.

The top half therefore contained immature, maturing as well as mature internodes

(approximately internodes 4 to 22) and the bottom half only mature internodes

(approximately internodes 23 to 39).

RESULTS AND DISCUSSION

Molecular characterisation of transgenic plants

The presence of the transgenes in the regenerated sugarcane plants was confirmed by

PCR analyses during the tissue culture stage. Confirmed transgenic lines, from which a

clear transgene fragment was amplified, were hardened off and grown under greenhouse

conditions. No obvious phenotypic differences were apparent between the wild type

control plants and the transgenic lines. Transgene expression was confirmed by northern

blot analyses in approximately 10 month old cane (Figure 1). Because only 53% of the

sugarcane PFP-β gene was used to construct the expression vectors it was easy to

differentiate between the endogenous messenger (2.3 kb) and the transgene messengers

(1.2 kb). The northern blot analyses did not only confirm the expression of the

transgenes in the different transgenic lines but also within the different tissues that were

analysed, i.e. leaf roll, immature and maturing internodes. In addition, a reduction of the

relative amount of the endogenous transcript was evident in the transgenic tissues. Only

lines in which the expression of the transgene was confirmed were used in subsequent

analyses. Lines transformed with an untranslatable form of the PFP-β were enumerated

with a clone number alone, e.g. 501-508, and lines transformed with an antisense

construct were enumerated with a Q and a clone number, e.g. Q3.

- 86 -

Figure 1. Northern blot analysis confirming the expression of the PFP-β transgene. Lanes (1) NCo310 (wild type control) leaf roll sample, (2) line 503 leaf roll sample, (3-5) line 502 leaf roll, internodes 3 and 5 samples respectively. The endogenous transcript is 2.3 kb while the untranslatable transgenic transcript is only 1.2 kb. The bottom gel represents the ethidium bromide stained ribosomal subunits that were used to verify equal loading.

Reduction in PFP activity

Expression of the antisense or untranslatable forms of the PFP-β gene resulted in a

significant reduction in PFP activity in internodal tissues (Figure 2). The extent to

which PFP activity was reduced in the internodal tissues of the transgenic plants was

depended on the developmental stage / maturity of the internode. In very young,

immature internodes, i.e. internodes 2-4 that had high PFP activity in the wild type, the

activity was reduced by up to 70%. In mature internodes, i.e. internodes ≥10 that have

low PFP activity in the wild type, activity was reduced to undetectable levels (Figure 3).

This pattern of reduced activity, i.e. more in mature than immature tissues, is similar to

that reported for tobacco sink (immature tissue) and source (mature tissue) leaves which

were engineered in the same way (Paul et al. 1995).

2.3 kb

1.2 kb

1 2 3 4 5

2.3 kb

1.2 kb

1 2 3 4 5

- 87 -

Figure 2. PFP activity in maturing internodal tissue, i.e. pooled internode 9 and 10 tissue of the wild type (WT), an untransformed tissue culture control (TC) and the various transgenic genotypes. Three replicate samples were prepared for each line.

Figure 3. PFP activity in different internodal tissues of wild type (-●-) and five representative transgenic genotypes: 501 (-◇-), 503 (-x-), 506 (-○-), Q3 (-△-) and Q4 (-□ -). The values reported are the means of three separate samples.

The reduction in PFP activity was accompanied by a similar decrease in the amount of

PFP-β protein as determined by protein blotting (data not shown). This concomitant

reduction in the amount of PFP-β protein and extractable PFP activity is consistent with

the work in potato (Hajirezaei et al. 1994) and tobacco (Paul et al. 1995) in which

antisense and co-suppression was also used to induce gene silencing. In combination,

this data suggest that the efficiency of antisense and co-suppression technology is

limited in tissues that express the endogenous gene at high levels. More recent advances

in RNA silencing (RNAi) technology (see Watson et al. 2005 for a review) could

therefore aid in down-regulating PFP activity more efficiently in young, metabolically

active tissues.

0

10

20

30

40

50

WT TC Q4 503 505 507 508 502 504 501 506 Q3

PFP

activ

ity

(nm

olm

in-1

mg-1

prot

ein)

0

10

20

30

40

50

WT TC Q4 503 505 507 508 502 504 501 506 Q3

PFP

activ

ity

(nm

olm

in-1

mg-1

prot

ein)

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

4 5 6 7 8 9 10 11 12 13Internode number

PFP

activ

ity

(nm

olm

in-1

mg-1

prot

ein)

0.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

4 5 6 7 8 9 10 11 12 13Internode number

PFP

activ

ity

(nm

olm

in-1

mg-1

prot

ein)

- 88 -

Influence of reduced PFP activity on sugar yields in greenhouse grown plants

To investigate the potential role of PFP in sucrose accumulation, sugars were extracted

from internodal tissue of greenhouse grown control, i.e. wild type NCo310, an

untransformed tissue culture control and transgenic plants and were quantified. Tissues

from internodes 3-5 were combined to represent immature internodes and that of

internode 12-14 to represent mature tissue. Sugar concentrations were variable when

expressed on a fresh weight basis and in comparison to the wild type the immature

tissue of the untransformed, tissue culture control had significantly higher hexose and

sucrose concentrations, which confounded the interpretation of these data (Figure 4).

Figure 4. Sugar concentrations in internode 3-5 (A) and 12-14 (B) tissue expressed as a function of fresh weight. Glucose (- -), fructose (- -) and sucrose (- -) concentrations were determined in triplicate samples; * indicates sucrose concentrations significantly (p < 0.05) different from that of the wild type.

The transgenic lines 506 and 507 had higher hexose concentrations in both the

immature and mature tissues, resulting in a significant (p < 0.05) reduction in purity, i.e.

sucrose concentration expressed as a percentage of the total sugar concentration, in both

the tissue types of these lines. In contrast, Q3 and Q4 had significantly lower hexose

concentrations in their immature tissues. Three transgenic lines, i.e. 501, Q3 and Q4,

0

100

200

300

400

500

600

700

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

200

400

600

800

1000

1200

1400A.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

FW)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

FW)

* *

* *

0

10

20

30

40

50

60

70

80

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

500

1000

1500

2000

2500

3000B.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

FW)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

FW)

*

*

**

0

100

200

300

400

500

600

700

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

200

400

600

800

1000

1200

1400A.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

FW)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

FW)

* *

* *

0

100

200

300

400

500

600

700

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

200

400

600

800

1000

1200

1400A.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

FW)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

FW)

* *

* *

0

10

20

30

40

50

60

70

80

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

500

1000

1500

2000

2500

3000B.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

FW)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

FW)

*

*

**

0

10

20

30

40

50

60

70

80

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

500

1000

1500

2000

2500

3000B.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

FW)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

FW)

*

*

**

- 89 -

had significantly higher sucrose concentrations in both their immature and mature

tissues (Figure 4), which translated into a significant increase in purity in the immature

tissues for the latter two lines. The variability in the fresh weight data makes the

comparison and interpretation of these results impossible. The most important factor

contributing to this variability is probably the watering regime used in the greenhouse

during the time of harvest. Expressing the data on a dry weight basis might therefore

allow a more standardised comparison.

In doing so, significant changes were evident in both the hexose and sucrose

concentrations in the immature tissues of most of the transgenic lines, while very few

significant differences were apparent in the mature tissues (Figure 5). These induced

changes were, however, not consistent, e.g. while most lines had unchanged or

increased hexose concentrations the two antisense lines, Q3 and Q4, had significantly

lower concentrations than the control lines. Similarly, lines 506 and 507 were the only

lines showing an increase in hexose concentrations in mature tissue. The variability

could be ascribed to factors as widely different as clonal/transformation effects

(undefined) to the physiological state of the cane at harvest (e.g. induced ripening could

lead to low hexose concentrations and vise versa) and compartmentalisation effects (e.g.

a large percentage of the sugars are localised in the vacuole and could therefore be

influenced by other enzyme activities not localised in the same compartment as PFP).

Although the glucose vs. fructose levels were similar in all the lines and tissue types, the

ratios between the hexose and sucrose concentrations were highly variable, especially in

the immature tissues (Figure 5A). Lines with the lowest sucrose concentrations had the

highest hexose concentrations and vice versa. Fifty percent of the transgenic lines had a

significantly higher sucrose concentration than the control in the immature tissues,

which translated into a significant (p < 0.05) increase in purity in these lines. In

contrast, the only significant change in the sucrose concentrations of the mature tissues

was a decrease in that of line Q3 (Figure 5B). In addition, two lines, i.e. 506 and 507,

also showed a significant increase in hexose concentrations and consequently, only

these two lines displayed a significant decrease in purity. The only changes that were

- 90 -

consistent between the fresh and dry weight data were the increase in sucrose

concentrations in the immature tissues of lines Q3 and Q4 and the high hexose

concentrations in lines 506 and 507.

Figure 5. Sugar concentrations in internode 3-5 (A) and 12-14 (B) tissue expressed as a function of dry weight. Glucose (- -), fructose (- -) and sucrose (- -) concentrations were determined in triplicate samples; * indicates sucrose concentrations significantly (p < 0.05) different from that of the wild type.

Correlation between PFP activity and sucrose yields

PFP activity and sucrose concentrations in immature and mature tissues from a

representative number of transgenic lines were correlated. Significant (p < 0.05) inverse

correlations were evident from the results when sucrose concentrations were expressed

on a fresh weight and protein basis (Figure 6A and C). This inverse correlation between

PFP activity and sucrose yields in the transgenic clones is consistent with that reported

in commercial varieties and a segregating F1 population (Whittaker and Botha 1999),

further supporting the apparent role for PFP in the sucrose accumulation phenotype in

sugarcane.

*

A.

B.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

0

100

200

300

400

500

600

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

500

1000

1500

2000

2500

3000

3500

4000

Hex

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

0

50

100

150

200

250

300

350

400

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

1000

2000

3000

4000

5000

6000

7000

*

* *

*

**

*

*

A.

B.

Hex

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

0

100

200

300

400

500

600

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

500

1000

1500

2000

2500

3000

3500

4000

Hex

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

Genotype

Sucr

ose

conc

entr

atio

n(µ

mol

g-1

DW

)

0

50

100

150

200

250

300

350

400

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

1000

2000

3000

4000

5000

6000

7000

0

50

100

150

200

250

300

350

400

WT TC 501 502 503 504 505 506 507 508 Q3 Q40

1000

2000

3000

4000

5000

6000

7000

*

* *

*

**

*

- 91 -

Figure 6. The relationship between sucrose yields and PFP activity in transgenic and control sugarcane lines. Solid and hollow symbols represent internode 3-5 and 12-14 tissues respectively and the wild type is represented by a square.

Influence of reduced PFP activity on sugar yields in field grown plants

To further investigate the influence of reduced PFP activity on sucrose yields, sets of

the greenhouse grown plants were planted in the field and analysed as described in the

materials and methods section. When the plant crop was harvested after approximately

15 months and the stalks were analysed as a whole no significant differences were

evident (data not shown). The plants were therefore allowed to ratoon and were again

harvested after 16 months. Based on the apparent differences between immature and

mature tissues of the greenhouse grown plants, the stalk samples were divided into a top

and a bottom half and analysed separately.

All the transgenic lines except 507 showed a significant decrease in total sugar yield in

both the top and bottom halves of the stalks (Figure 7A). This was true when total sugar

yield was expressed on a dry weight as well as a fresh weight basis. Moreover, the

relative differences between the various tissues and lines were exactly the same when

expressed either on a dry weight or a fresh weight basis. In other words, in contrast to

A.r2 = 0.378

0500

10001500200025003000

0 20 40 60 80 100 120 140 160PFP activity (nmol min-1 mg-1 protein)

Sucr

ose

(µm

ol g

-1FW

)

r2 = 0.305

0100020003000400050006000

0 50 100 150 200PFP activity (nmol min-1 mg-1 protein)

Sucr

ose

(µm

ol g

-1D

W)

r2 = 0.416

0

5001000

1500

2000


Sucr

ose

(µm

ol m

g-1pr

otei

n)

B.

C.

p = 0.033

p = 0.062

p = 0.023

A.r2 = 0.378

0500

10001500200025003000


Sucr

ose

(µm

ol g

-1FW

)

r2 = 0.305

0100020003000400050006000


Sucr

ose

(µm

ol g

-1D

W)

r2 = 0.416

0

5001000

1500

2000


Sucr

ose

(µm

ol m

g-1pr

otei

n)

B.

C.

p = 0.033

p = 0.062

p = 0.023

r2 = 0.378

0500

10001500200025003000


Sucr

ose

(µm

ol g

-1FW

)

r2 = 0.305

0100020003000400050006000


Sucr

ose

(µm

ol g

-1D

W)

r2 = 0.416

0

5001000

1500

2000


Sucr

ose

(µm

ol m

g-1pr

otei

n)

B.

C.

p = 0.033

p = 0.062

p = 0.023

- 92 -

the greenhouse data, there were no differences between the fresh and dry weight data of

the field trial. This could possibly be attributed to the fact that all the plants were well

established at the time of the first ratoon and that they were all exposed to exactly the

same growing conditions.

Figure 7. Sugar data for field grown wild type (WT) and transgenic sugarcane lines with reduced PFP activity. The harvested cane was divided into the top (- -) and bottom (- -) halves and analysed for (A) Brix % DW, (B) Pol % cane and (C) % purity. The data was generated from triplicate samples; * indicates measurements significantly (p < 0.05) different from that of the respective tissues of the wild type.

In contrast to the total sugar yield, sucrose yields expressed on a fresh weight basis (Pol

% cane) significantly increased only in the top halves of five of the transgenic lines

(Figure 7B). Although the bottom half of several lines, i.e. 503, 504, 505 and 507, had

up to 5% higher average sucrose yields than the wild type, this was not significant. In

Genotype

Bri

x%

DW

0

10

20

30

40

50

60

70

80

WT 501 502 503 504 505 506 507 Q3 Q4

*** ** ** ** **

** **

Genotype

% P

urity

0102030405060708090

100

WT 501 502 503 504 505 506 507 Q3 Q4

** ** ** ** ** **

** **C.

B.

A.

Genotype

Pol%

can

e

02468

101214161820

WT 501 502 503 504 505 506 507 Q3 Q4

*** * **

Genotype

Bri

x%

DW

0

10

20

30

40

50

60

70

80

WT 501 502 503 504 505 506 507 Q3 Q4

*** ** ** ** **

** **

Genotype

Bri

x%

DW

0

10

20

30

40

50

60

70

80

WT 501 502 503 504 505 506 507 Q3 Q4

*** ** ** ** ** ** ** ** ** **

** ** ** **

Genotype

% P

urity

0102030405060708090

100

WT 501 502 503 504 505 506 507 Q3 Q4

** ** ** ** ** **

** **

Genotype

% P

urity

0102030405060708090

100

WT 501 502 503 504 505 506 507 Q3 Q4

** ** ** ** ** ** ** ** ** ** ** **

** ** ** **C.

B.

A.

Genotype

Pol%

can

e

02468

101214161820

WT 501 502 503 504 505 506 507 Q3 Q4

*** * **

Genotype

Pol%

can

e

02468

101214161820

WT 501 502 503 504 505 506 507 Q3 Q4

** *** * **

- 93 -

fact, the only significant difference in sucrose yields in the bottom half of the stalks was

a slight decrease in yield for line Q3 (Figure 7B). Although all except one transgenic

line had a higher average sucrose yield than the wild type when calculated for the whole

stalk, these increases were not significant (data not shown). The increase in sucrose

yields in immature / maturing internodes was therefore consistent between the

greenhouse and field grown material. Although the increase seemed less pronounced in

the field grown tissue; a maximum of 20% vs. 130% in the greenhouse, this was

expected because the field grown material represented the average of more mature

tissue, i.e. approximately internode 4-22, while the greenhouse immature samples only

represented the very immature internodes 3-5. In addition, as described in the materials

and methods section, the greenhouse samples represented only core tissue,

predominantly storage parenchyma cells, while the field samples also included the rind

and nodal tissue which do not store sucrose at high concentrations.

Similar to the total sugar data the purity of all the transgenic lines, bar 507, for both

tissue types changed significantly, but in contrast to the total sugar content the purity

increased (Figure 7C). Moreover, in all cases the decrease in total sugar yield correlated

with a similar increase in purity. In combination with the constant or slight increase in

sucrose concentrations it therefore suggests that hexose concentrations were

significantly reduced in most transgenic lines. A reduction in hexose concentrations can

be attributed to a reduction in the total sucrose breakdown activity or an increased flux

out of the hexose pool or a combination thereof. Furthermore, if these changes can be

ascribed to the reduction in PFP activity it either implies that it is the cytosolic hexose

concentrations that are influenced or that the cytosolic and vacuolar hexose pools are in

a dynamic equilibrium. The consistency of the data between the immature and mature

tissues also indirectly supports this conclusion because the increased contribution of the

vacuolar constituents should influence the changes non-symmetrically as the tissues

mature.

Reduction in PFP activity leads to an increase in the hexose phosphate pool in sink

tissues (Hajirezaei et al. 1994, Paul et al. 1995), which could lead to a decrease in the

- 94 -

rate of sucrose breakdown (Geigenberger et al. 1994, Hajirezaei et al. 1994). Under

these conditions sucrose synthesis by sucrose phosphate synthase (SPS) will also be

stimulated because of the increased levels of glucose 6-phosphate (Glc 6-P), a strong

allosteric activator of SPS (Reimholz et al. 1994), and SPS’s substrates (Dancer et al.

1990, Hajirezaei et al. 1994). Hajirezaei et al. (1994) also showed that a decrease in

PFP activity leads to an increased flux of glucose into sucrose; although the hexose

concentrations in these plants are highly variable in all the tissue types that were

analysed. The validity of these arguments for the sugarcane plants with reduced PFP

activity can only be verified by determining the actual flux into sucrose.

Fibre content was also significantly increased in both the tissue types in most of the

transgenic lines (Figure 8). The fibre data mirrored the purity data, which also means

that the differences, relative to the wild type, correlated inversely to those observed for

the total sugars (Brix %, Figure 7A). In other words, a large decrease in total sugar

content relative to the wild type, correlated with a large increase in the fibre content and

a small decrease in sugar with a small increase in fibre. Again, this could be explained

by an increase in the hexose phosphate pool, which could lead to an increase in the cell

wall precursors via UDP-glucose. UDP-glucose and the hexose phosphate pool are

connected by a series of highly active, reversible reactions, which should ensure

equilibrium between these metabolites (ap Rees 1988, Tobias et al. 1992). Because the

millroom data are expressed as percentages the possibility that the said reduction in total

sugar yield is due to a corresponding increase in fibre content cannot be excluded and

should be further investigated.

- 95 -

Figure 8. Fibre content of field grown wild type (WT) and transgenic lines with reduced PFP activity. The harvested cane was divided into the top (- -) and bottom (- -) halves, analysed for fibre content and expressed on a fresh weight basis (% cane). The data was generated from triplicate samples; * indicates measurements significantly (p < 0.05) different from that of the respective tissues of the wild type.

Cumulatively the field data confirms that reduced PFP activity influences sugar

metabolism in sugarcane internodal tissues. Similar to the greenhouse grown plants, the

effect on sucrose accumulation was the greatest in the more immature, metabolically

active tissues even though PFP activity was only down-regulated to 30% of that of the

wild type in these tissues. This apparent difference with which the different tissues

respond to decreased PFP activity is similar to that reported for growing (sink) and

sprouting (source) potato tubers (Hajirezaei et al. 1994) and growing (sink) tobacco

leaves (Nielsen and Stitt 2001) in contrast to mature tubers and leaves. The determining

parameter therefore seems not to be the absolute change in PFP activity in a particular

tissue but rather the relative contribution PFP makes to the glycolytic flux in that tissue.

In immature internodal tissues the reduction in PFP activity was sufficient to impede on

the high requirement for respiratory flux, probably resulting in the accumulation of the

hexose phosphates as observed in similar studies (Hajirezaei et al. 1994, Paul et al.

1995). Elevated hexose phosphate levels in turn could result in increased sucrose

synthesis and storage as well as elevated cell wall synthesis as discussed above. As the

internodes mature and the demand for respiratory flux decreases (Bindon and Botha

2002) the residual PFP activity and the alternative reaction catalysed by PFK is

sufficient and the system returns to equilibrium, ending with sugar levels comparable to

that of the unimpeded system. It is important to note that although sucrose, a part of

**

02

468

10

121416

1820

WT 501 502 503 504 505 506 507 Q3 Q4

Genotype

Fibr

e %

can

e

**** ** ** *

****

** **

02

468

10

121416

1820

WT 501 502 503 504 505 506 507 Q3 Q4

Genotype

Fibr

e %

can

e

** **** ** ** ** ** ** *

** **** **

- 96 -

dynamic carbon metabolism, returned to equilibrium in the older internodes the amount

of fibre in the mature internodes were still significantly higher in most transgenic lines –

probably because most of the carbon is fixed during the developmental stage most

impeded upon by the reduced levels of PFP. The data generated with the transgenic

sugarcane lines, similar to the studies in potato and tobacco (Hajirezaei et al. 1994, Paul

et al. 1995), supports the suggested role of PFP as a bypass to PFK at times of high

metabolic flux in biosynthetically active tissue as suggested by Dennis and Greyson

(1987).

To confirm these speculative statements the net glycolytic fluxes and detailed metabolic

analyses should be performed on these transgenic lines. In addition, determining the

extent of the triose-hexose phosphate cycle, especially in mature internodes, could shed

some light on the presence of FBPase in non-photosynthetic tissues. Finally, the more

efficient down-regulation of PFP activity, particularly in immature tissues, should be

attempted using RNAi technology. Al these aspects are currently under investigation in

our laboratory.

CONCLUSION

The down-regulation of PFP activity in sugarcane confirmed that it is inversely

correlated to the sucrose accumulation phenotype, but more specifically only in

metabolically active, immature tissues. Moreover, it suggests that PFP plays a

significant role in glycolytic carbon flux in immature, metabolically active sugarcane

internodal tissues. The data presented here confirm that PFP can indeed have an

influence on the rate of glycolysis and carbon partitioning in these tissues

ACKNOWLEDGEMENTS

The South African Sugar Association, the South African Department of Trade and

Industry and Stellenbosch University sponsored this work.

- 97 -

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Sung SS, Xu D-P, and Black CC (1988) Identification of actively filling sucrose sinks. Plant Physiol 89: 1117-1121.

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Theodorou ME, Plaxton WC (1996) Purification and characterisation of Pyrophosphate-dependent Phosphofructokinase from phosphate-starved Brassica nigra suspension cells. Plant Physiol 112: 343-351

Tobias RB, Boyer CD, Shannon JC (1992) Enzymes catalyzing the reversible conversion of fructose-phosphate and fructose-1,6-bisphosphate in maize (Zea mays L) kernels. Plant Physiol 99: 140-145

van Schaftingen E, Lederer B, Bartons R, Hers H-G (1982) A kinetic study of pyrophosphate: fructose-6-phosphate phosphotransferase from potato tubers. European Journal of Biochemistry 129: 191-195

Watson JM, Fusaro AF, Wang MB, Waterhouse PM (2005) RNA silencing platforms in plants. FEBS Lett 579: 5982-5987

Weiner H, Stitt M, and Heldt HW (1987) Subcellular compartmentation of pyrophosphate and pyrophosphatase. Biochim.Biophys.Acta 893: 13-21.

Whittaker A (1997) Pyrophosphate dependent phosphofructokinase (PFP) activity and other aspects of sucrose metabolism in sugarcane internodal tissues. PhD thesis, University of Natal, South Africa pp 1-187

Whittaker A, Botha FC (1997) Carbon partitioning during sucrose accumulation in sugarcane internodal tissue. Plant Physiology 115: 1651-1659


Wong JH, Kang T, Buchanan BB (1988) A novel pyrophosphate fructose-6-phosphate 1-phosphotransferase from carrot roots. Relation to PFK from the same source. FEBS Lett 238: 405-410

Wong JH, Kiss F, Wu M-X, Buchanan BB (1990) Pyrophosphate Fructose 6-P 1-Phosphotransferase from tomato fruit. Plant Physiol 94: 499-506

Wood SM, Newcomb W, Dennis DT (2002a) Overexpression of the glycolytic enzyme Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) in developing transgenic tobacco seeds results in alterationsin the onset and extent of storage lipid deposition. Can J Bot 80: 993-1001

Wood SM, King SP, Kuzma MM, Blakeley SD, Newcomb W, Dennis DT (2002b) Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase overexpression in transgenic tobacco: physiological and biochemical analysis of source and sink tissues. Can J Bot 80: 983-992

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CHAPTER 6

General discussion and conclusions

The main aim of the work presented in this thesis was to elucidate the apparent role of

pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose

accumulation in sugarcane. PFP activity in sugarcane internodal tissue is inversely

correlated to the sucrose content and positively to the water-insoluble component across

varieties which differ in their capacities to accumulate sucrose (Whittaker and Botha

1999). This apparent well defined and important role of PFP seems to stand in contrast

to the ambiguity regarding PFP’s role in the general literature as well as the results of

various transgenic studies where neither the down-regulation (Hajirezaei et al. 1994,

Paul et al. 1995, Nielsen and Stitt 2001) nor the over-expression (Wood et al. 2002a,

2002b) of PFP activity had a major influence on the phenotype of the transgenic plants.

Based on this it was therefore thought that either the kinetic properties of sugarcane PFP

are significantly different than that of other plant PFPs or that PFP’s role in sucrose

accumulating tissues is different from that in starch accumulating tissues – clearly an

issue that needed resolution.

If there is indeed a negative correlation between PFP expression and sucrose levels in

sugarcane, two different probable explanations for this can be offered based on current

literature. Firstly, it could be a direct consequence of a reduction in glycolytic carbon

flow through the PFP-catalysed reaction and the subsequent alteration in carbon

partitioning. Moreover, if the PFP-catalysed reaction is rate limiting in sucrose

accumulating tissues, decreased activity should result in an increase in the hexose-

phosphate pool, which could lead to increased sucrose synthesis and subsequent storage.

This model is consistent with the general consensus that PFP catalyses a net glycolytic

flux (see Stitt 1990 for review), which is also true for sugarcane (Whittaker and Botha

1999). Secondly, the effect might be linked to the presence of a H+-sucrose antiport

system in the tonoplast, which could be more effectively energised if the activity of a

H+-translocating vacuolar pyrophosphatase (VPPase) is enhanced. This could happen in

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an environment where low PFP activity translates into the reduced utilisation of PPi, but

because PPi concentrations need to be finely regulated (Weiner et al. 1987, Takeshige

and Tazawa 1989) at least some of this burden could be transferred to VPPase, resulting

in the enhanced energisation of the tonoplast. This could be of particular importance in

metabolically active tissues where many biosynthetic reactions produce PPi as a by-

product and their continuation is dependent on the effective removal of the PPi.

Reduced glycolytic carbon flow through the PFP-catalysed reaction could be the result

of reduced total activity or adapted kinetic characteristics, which could for example

favour the gluconeogenic reaction and in doing so, increase the concentrations of the

precursors for sucrose synthesis as suggested by Wong et al. (1988, 1990) for other

sugar storing tissues. In addition, albeit indirect, support for the apparent importance of

the gluconeogenic reaction in sucrose storing tissues is also provided by the unique

characteristics of grapefruit FBPase. Grapefruit juice sac FBPase has a much higher (up

to 20x) affinity for Fru 1,6-P2 than other plant FBPases, which might be an adaptation

for sucrose biosynthesis in sink tissues (Van Praag 1997). However, we have shown in

Chapter 3 that sugarcane PFP’s molecular and kinetic properties do not differ

significantly from that of other plant PFPs in a way that clearly suggests a direct role for

it in the accumulation of sucrose. If the apparent link between PFP activity and sucrose

accumulation is based on flux through this reaction it is therefore probably based on the

total amount of catalytic activity present in the tissue rather than the fine regulation

thereof. This conclusion is also consistent with the coarse regulation of activity in these

tissues as illustrated by Whittaker and Botha (1999) as well as the suggestion that the

total amount of PFP activity in sugarcane is influenced more by genotype than

developmental and/or fine regulatory factors (Chapter 3); analogous to sucrose storing

carrot suspension cells (Krook et al. 2000). If this is indeed the case it suggests great

promise for the transgenic down-regulation of PFP activity in sugarcane.

PFP activity was successfully down-regulated in transgenic sugarcane (Chapter 5). The

degree to which PFP activity was reduced varied between different tissue types,

depending on the basal endogenous activity and overall metabolic activity in the

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particular tissue. These findings are similar to that reported for tobacco sink (immature

tissue) and source (mature tissue) leaves which were engineered in the same way (Paul

et al. 1995). In general sugar metabolism was impeded upon most in young,

metabolically active internodal tissues resulting in an “early ripening” phenotype, i.e.

increased sucrose concentrations and higher purity in immature internodes. No

consistent differences were apparent in the sugar concentrations of the mature

internodes. This was true even though, if expressed as percentage reduction in activity,

PFP activity was down-regulated to a lesser extent in the immature than in the mature

internodes.

The influence of PFP on the metabolism of these tissues therefore seems to depend on

the total catalytic activity (glycolytic flux) required and PFP’s relative contribution to

this flux rather than an exclusive function for PFP in sucrose accumulation. Another

way of interpreting this is that the influence of reduced PFP activity is of a transient

nature, i.e. as the effected tissues mature equilibrium is restored, even in the apparent

absence of PFP activity (Figure 3, Chapter 5). As the transgenic cells mature and the

required glycolytic flux decreases to levels which can be sustained by PFK and the

residual PFP, the equilibrium of the glycolytic intermediates, including the stored

sucrose, is restored. PFP therefore does not influence the overall ability of the sugarcane

cells to accumulate sucrose but can influence the rate at which it happens in tissues with

a high glycolytic flux. This explains why sucrose yields were significantly increased in

immature internodes but not in mature internodes.

Commercial sugarcane plants are up to three meters tall, consisting of approximately 40

internodes, and are grown over 12 to 24 months. The proportion of immature tissue to

mature tissue in the stalk at the time of harvest is small (less than 5% immature tissue).

The dynamic metabolite pools in the bulk of the transgenic tissues therefore have ample

opportunity to return to equilibrium, resulting in total sucrose yields similar to that of

the wild type. Notwithstanding, as has been illustrated by the use of commercial

ripeners this “early ripening” phenotype could contribute to the overall productivity of

the crop if the right varieties are targeted. In addition, in the light of the very

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discouraging yield data of a field trial with transgenic sugarcane in Australia, where

98% of the lines had a reduced sucrose content (Vickers et al. 2005), the data presented

here are indeed promising.

All the above mentioned arguments agree with the original data that suggested a role for

PFP in sucrose accumulation (Whittaker and Botha 1999). Moreover, the fact that these

data were also obtained from metabolically active tissues (internode seven) with a high

sucrose accumulation rate fits well with the findings of this study. Considering the

above mentioned ability of the sucrose pool to return to equilibrium over time, it implies

that PFP does not play a role in determining the maximum sucrose load in mature

tissues.

As discussed in Chapter 5, a reduction in the glycolytic carbon flow due to low PFP

activity should lead to increased levels of hexose phosphate pool, including UDP-

glucose and consequently increased cell wall/cellulose synthesis (Fig. 1, Chapter 1) (ap

Rees 1988, Tobias et al. 1992). In the transgenic plants the most active stage of cell wall

synthesis therefore coincides with the stage most impeded upon by the reduced levels of

PFP, resulting in tissues with significantly higher fibre than the wild type (Fig. 7,

Chapter 5). In contrast to the glycolytic intermediates the carbon fixed during cell wall

synthesis, which will form part of the water-insoluble component, can not be

remobilised and any quantitative changes should therefore remain in the mature

internodes, resulting in an increase in fibre content.

It is interesting to note that the water-insoluble component is positively correlated to

PFP activity across commercial varieties (Whittaker and Botha 1999), whereas reduced

PFP activity in the transgenic plants had a similar effect (Fig. 7, Chapter 5). The reason

for this apparent discrepancy probably lies in the diverse composition of the water-

insoluble component. One plausible explanation is that in tissues with high PFP activity

it contributes to an increased total respiratory flux which could translate into a water-

insoluble component with increased protein content (Bindon and Botha 2002). In the

transgenic plants, on the other hand, reduced PFP activity results in an increase in the

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water-insoluble component by increasing cellulose synthesis as explained above. PFP

could also influence the insoluble component through its effect on fatty acid

metabolism. PFP is for example implicated in the inability of wrinkled1 mutant

Arabidopsis seeds to accumulate triacylglycerol (Focks and Benning 1998) and also

influences lipid content and the onset of lipid deposition in transgenic tobacco seeds

(Wood et al. 2002a, 2002b).

The most obvious priority for future work is the comprehensive metabolic analysis of

the transgenic plants. Potential changes in the levels of metabolites directly associated

with PFP activity should be determined to confirm for example the reduction in

glycolytic flux. Other aspects that need to be investigated include: (i) Determining the

extent to which the triose-phosphate : hexose-phosphate cycle was influenced. (ii)

Quantifying the possible change in total respiration. (iii) Qualifying potential changes in

the water-insoluble component. Research priorities aimed at commercial application

could include the more efficient reduction of PFP activity in the immature tissues using

RNAi and the down-regulation of both PFP and UDPGlc-DH activity in transgenic

plants.

With respect to the proposed second hypothesis in Chapter 2 the potential influence of

reduced PFP activity on PPi metabolism should be investigated, with special emphasis

on possible changes in VPPase activity in the transgenic plants. This line of

investigation should be a priority because of the important insight gained in this study

into the dynamic nature of the sucrose pool, i.e. its ability to return to equilibrium over

time. Enhanced sucrose transport out of the metabolically active compartment into the

vacuole might therefore represent a viable system to increase the maximum sucrose

content in sugarcane tissues. This could also be interpreted as a terminal reaction for

sucrose accumulation, which in general is a good target for manipulating the flux

through a particular metabolic network (see Kinney 1998 for a review).

In conclusion, this study confirmed the negative correlation between PFP activity and

sucrose content of immature sugarcane internodal tissue. Moreover, it suggests that PFP

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plays a significant role in glycolytic carbon flux in immature, metabolically active

sugarcane internodal tissues. The data presented here confirm that PFP can indeed have

an influence on the rate of glycolysis and carbon partitioning in these tissues. Although

this seems to be in disagreement with the general conclusions of the first transgenic

studies that were published (Hajirezaei et al. 1994, Paul et al. 1995) the data on

immature tissues in these papers and the subsequent work (Nielsen and Stitt 2001,

Wood et al. 2002a) support this conclusion. It therefore also implies that there are no

differences between the functions of PFP in starch and sucrose storing tissues and it

supports the hypothesis that PFP provides additional capacity to PFK at times of high

metabolic flux in biosynthetically active tissue (Dennis and Greyson 1987).

REFERENCES

ap Rees T (1988) Hexose phosphate metabolism by non-photosynthetic tissues of higher plants. In J Preiss, ed Biochemistry of plants Vol 14. Academic Press, New York, pp 1-33


Dennis DT, Greyson MF (1987) Fructose 6-phosphate metabolism in plants. Physiologia Plantarum 69: 395-404

Focks N, Benning C (1998) wrinkled1: A Novel, low-seed-oil mutant of Arabidopsis with a deficiency in the seed-specific regulation of carbohydrate metabolism. Plant Physiology 118: 91-101


Kinney AJ (1998) Manipulating flux through plant metabolic pathways. Current Opinion in Plant Biology 1: 173-178

Krook J, Van't Slot KAE, Vruegdenhil D, Dijkema C, van der Plas L (2000) The triose-hexose phosphate cycle and the sucrose cycle in carrot (Daucus carota L.) cell suspensions are controlled by respiration and PPi: Fructose-6-phosphate phosphotransferase. Plant Physiol 156: 595-604

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Nielsen TH, Stitt M (2001) Tobacco transformants with strongly decreased expression of pyrophosphate: fructose-6-phosphate expression in the base of their young growing leaves contain much higher levels of fructose-2,6-bisphosphate but no major changes in fluxes. Planta 214: 106-116



Takeshige K, Tazawa M (1989) Determination of the inorganic pyrophosphatase level and its subcellular localization in Chara corallina. J. Biol. Chem. 264: 3262-3266.

Tobias RB, Boyer CD, Shannon JC (1992) Enzymes catalyzing the reversible conversion of fructose-phosphate and fructose-1,6-bisphosphate in maize (Zea mays L) kernels. Plant Physiol 99: 140-145

Van Praag E (1997) Kinetic properties of cytosolic fructose 1,6-bisphosphatase from grapefruit: Effect of citrate. Bioch Mol Biol Int 43: 625-631.

Vickers JE, Grof CPL, Bonnett GD, Jackson PA, Morgan TE (2005) Effects of tissue culture, biolistic transformation, and introduction of PPO and SPS gene constructs on performance of sugarcane in the field. Aust J Agr Res 56: 57-68

Weiner H, Stitt M, Heldt HW (1987) Subcellular compartmentation of pyrophosphate and pyrophosphatase. Biochim Biophys Acta 893: 13-21.


Wong JH, Kang T, Buchanan BB (1988) A novel pyrophosphate fructose-6-phosphate 1-phosphotransferase from carrot roots. Relation to PFK from the same source. FEBS Lett 238: 405-410

Wong JH, Kiss F, Wu M-X, Buchanan BB (1990) Pyrophosphate Fructose 6-P 1-Phosphotransferase from tomato fruit. Plant Physiol 94: 499-506

Wood SM, King SP, Kuzma MM, Blakeley SD, Newcomb W, Dennis DT (2002a) Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase overexpression in transgenic tobacco: physiological and biochemical analysis of source and sink tissues. Can J Bot 80: 983-992

- 107 -

Wood SM, Newcomb W, Dennis DT (2002b) Overexpression of the glycolytic enzyme Pyrophosphate-dependent fructose-6-phosphate 1-phosphotransferase (PFP) in developing transgenic tobacco seeds results in alterationsin the onset and extent of storage lipid deposition. Can J Bot 80: 993-1001

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Manipulation of pyrophosphate fructose 6-phosphate 1